分子生物學

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(1)分子生物學. 生物科學系陳錦翠.

(2) THE MOLECULE BASIS OF INHERITANCE DNA as the Genetic Material. 1953, James Watson and Francis Crick 1962 Nobel prize.

(3) The search for genetic material lead to DNA • Once T.H. Morgan’s group showed that genes are located on chromosomes, the two constituents of chromosomes proteins and DNA - were the candidates for the genetic material. • Until the 1940s, the great heterogeneity and specificity of function of proteins seemed to indicate that proteins were the genetic material. • However, this was not consistent with experiments with microorganisms, like bacteria and viruses..

(4) Frederick Griffith experiment in 1928 Living S cells (control). Living R cells (control). Heat-killed S cells (control). Mixture of heat-killed S cells and living R cells. RESULTS. Mouse dies. Mouse healthy. Mouse healthy. Frederick Griffith, 1928. Mouse dies. Living S cells.

(5) • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928. • Griffith called this phenomenon transformation(轉型), a change in genotype and phenotype due to the assimilation(同化) of a foreign substance (now known to be DNA) by a cell. • For the next 14 years scientists tried to identify the transforming substance. • Finally in 1944, Oswald Avery, Maclyn McCarty and Colin MacLeod announced that the transforming substance was DNA.

(6) • In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the bacteriophage T2. Cold Spring Harbor Laboratory. Department of Genetics.

(7) Hershey and Chase’s experiment.

(8) • By 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms. • He already knew that DNA was a polymer of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group. • The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C). • Chargaff noted that the DNA composition varies from species to species. • In any one species, the four bases are found in characteristic, but not necessarily equal, ratios..

(9) • He also found a peculiar regularity in the ratios of nucleotide bases which are known as Chargaff’s rules. • The number of adenines was approximately equal to the number of thymines (%T = %A). • The number of guanines was approximately equal to the number of cytosines (%G = %C). • Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine and 19.8% cytosine..

(10) Watson and Crick discovered the double helix by building models to conform to X-ray data. • By the beginnings of the 1950’s, the race was on to move from the structure of a single DNA strand to the threedimensional structure of DNA. • Among the scientists working on the problem were Linus Pauling, in California, Caltech Maurice Wilkins and Rosalind Franklin, in London..

(11) • The phosphate(磷酸) group of one nucleotide(核苷酸) is attached to the sugar of the next nucleotide in line. • The result is a “backbone” of alternating phosphates and sugars, from which the bases project..

(12) • Maurice Wilkins and Rosalind Franklin used X-ray crystallography (X射線晶體學) to study the structure of DNA. • In this technique, X-rays are diffracted as they passed through aligned fibers of purified DNA.. • The diffraction(繞射) pattern can be used to deduce the three-dimensional shape of molecules. • James Watson learned from their research that DNA was helical in shape and he deduced the width of the helix and the spacing of bases.. Rosalind Franklin died in the age of 38.

(13) • Watson and his colleague Francis Crick began to work on a model of DNA with two strands, the double helix (雙螺旋). • Using molecular models made of wire, they first tried to place the sugar-phosphate chains on the inside. • However, this did not fit the X-ray measurements and other information on the chemistry of DNA. • The key breakthrough came when Watson put the sugarphosphate chain on the outside and the nitrogen bases on the inside of the double helix. • The sugar-phosphate chains of each strand are like the side ropes of a rope ladder. • Pairs of nitrogen bases, one from each strand, form rungs. • The ladder forms a twist every ten bases..

(14) Watson built a model in which the backbones were antiparallel 反向平行 (their subunits run in opposite directions).

(15) • The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine. • Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data. • A purine-purine pair would be too wide and a pyrimidinepyrimidine pairing would be too short. • Only a pyrimidinepurine pairing would produce the 2-nm diameter indicated by the X-ray data..

(16) • In addition, Watson and Crick determined that chemical side groups of the nitrogen bases would form hydrogen bonds, connecting the two strands. • Based on details of their structure, adenine would form two hydrogen bonds only with thymine and guanine would form three hydrogen bonds only with cytosine.. 腺嘌呤胸腺嘧啶. • This finding explained Chargaff’s rules.. 鳥嘌呤. 胞嘧啶.

(17) • The base-pairing rules dictate the combinations of nitrogenous bases that form the “rungs” of DNA. • However, this does not restrict the sequence of nucleotides along each DNA strand. • The linear sequence of the four bases can be varied in countless ways. • Each gene has a unique order of nitrogen bases. • In April 1953, Watson and Crick published a succinct, onepage paper in Nature reporting their double helix model of DNA..

(18) DNA Replication.

(19) During DNA replication, base pairing enables existing DNA strands to serve as templates (模版) for new complimentary(互補) strands.

(20) • In a second paper Watson and Crick published their hypothesis for how DNA replicates • Watson and Crick’s model, semiconservative replication •. 半保留,半保守.

(21) • Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models..

(22) • In eukaryotes, there may be hundreds or thousands of origin sites (起始點) per chromosome. • At the origin sites, the DNA strands separate forming a replication “bubble” with replication forks at each end. 複製叉.

(23) (a) Origin of replication in an E. coli cell Origin of replication. (b) Origins of replication in a eukaryotic cell. Parental (template) strand. Origin of replication. Daughter (new) strand Doublestranded DNA molecule. Replication fork Replication bubble. Parental (template) strand. Bubble. Double-stranded DNA molecule. Daughter (new) strand. Replication fork. Two daughter DNA molecules. 0.25 µm. 0.5 µm. Two daughter DNA molecules.

(24) • DNA polymerases聚合酶 catalyze the elongation of new DNA at a replication fork. • As nucleotides align with complementary bases along the template strand, they are added to the growing end of the new strand by the polymerase. • The rate of elongation: is about 500 nucleotides per second in bacteria and 50 per second in human cells. The raw nucleotides are nucleoside triphosphates.. • The raw nucleotides are nucleoside triphosphates.核苷三磷酸 Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings.

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(26) • The strands in the double helix are antiparallel..

(27) • To summarize, at the replication fork, the leading strand is copied continuously into the fork from a single primer. • The lagging strand (先導股) is copied away from the fork in short segments, each requiring a new primer. Overview Leading strand. Origin of replication. Lagging strand. Lagging strand 2. 1. 延遲股. Overall directions of replication. Leading strand.

(28) A summary of bacterial DNA replication (複製). DNA pol III synthesizes leading strand continuously. Parental DNA. 3′ 5′. DNA pol III starts DNA synthesis at 3′ end of primer, continues in 5′ → 3′ direction. 5′ 3′ 5′ Helicase 解旋酶. Origin of replication. Lagging strand synthesized in short (100-200 nts) Okazaki fragments (岡崎片段), later joined by DNA ligase. 3′ 5′. Primase synthesizes a short RNA primer 引子 DNA pol I replaces the RNA primer with DNA nucleotides. 100-200 nucleotides.

(29) • DNA polymerases cannot initiate synthesis of a polynucleotide because they can only add nucleotides to the end of an existing chain that is base-paired with the template strand. • To start a new chain requires a primer, a short segment of RNA. • The primer is about 10 nucleotides long in eukaryotes. • Primase(引子酶), an RNA polymerase, links ribonucleotides that are complementary to the DNA template into the primer. • Primase starts an RNA chain from a single template strand..

(30) • DNA polymerase III then adds deoxyribonucleotides (去氧核糖核苷酸) to the 3’ end of the ribonucleotide chain. • Another DNA polymerase I later replaces the primer ribonucleotides with deoxyribonucleotides complimentary to the template. • After the primer is formed, DNA polymerase can add new nucleotides away from the fork until it runs into the previous Okazaki fragment. • The primers are converted to DNA before DNA ligase (連接酶) joins the fragments together..

(31) • Returning to the original problem at the replication fork, the leading strand requires the formation of only a single primer as the replication fork continues to separate. • The lagging strand requires formation of a new primer as the replication fork progresses. • In addition to primase, DNA polymerases, and DNA ligases, several other proteins have prominent roles in DNA synthesis. • A helicase (解旋酶) untwists and separates the template DNA strands at the replication fork..

(32) • Single-strand binding proteins keep the unpaired template strands apart during replication and protect the template from degradation..

(33) A current model of the DNA replication complex. DNA pol III Parental DNA 5′ 3′. 5′. 3′. 3′ 5′. 5′. Connecting protein. 3′. Helicase. 3′ DNA pol III 5′. Leading strand. 3′. 5′. Lagging strand. Lagging strand template.

(34) • It is conventional and convenient to think of the DNA polymerase molecules moving along a stationary DNA template. • In reality, the various proteins involved in DNA replication form a single large complex that may be anchored to the nuclear matrix (核基質). • The DNA polymerase molecules “reel in”(捲入) the parental DNA and “extrude” newly made daughter DNA molecules.. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings.

(35) Enzymes proofread DNA during its replication and repair damage in existing DNA • Mistakes during the initial pairing of template nucleotides and complementary nucleotides occurs at a rate of one error per 10,000 base pairs. • DNA polymerase proofreads (校正) each new nucleotide against the template nucleotide as soon as it is added. • If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis. • The final error rate is only one per billion (1/109) nucleotides.. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings.

(36) The ends of DNA molecules are replicated by a special mechanism. 5′ Leading strand Lagging strand. Ends of parental DNA strands 3′. Last fragment. Previous fragment. RNA primer. Lagging strand 5′ 3′ Parental strand. Removal of primers and replacement with DNA where a 3′ end is available. 5′ 3′ Second round of replication 5′ New leading strand 3′ New lagging strand 5′ 3′ Further rounds of replication Shorter and shorter daughter molecules.

(37) • The ends of eukaryotic chromosomal DNA molecules, the telomeres端粒, have special nucleotide sequences. • In human telomeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times.. • Telomeres protect genes from being eroded through multiple rounds of DNA replication.. Fig. 16.19a Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings.

(38) • Eukaryotic cells have evolved a mechanism to restore shortened telomeres. • Telomerase端粒酶uses a short molecule of RNA as a template to extend the 3’ end of the telomere. • There is now room for primase and DNA polymerase to extend the 5’ end. • It does not repair the 3’-end “overhang,” but it does lengthen the telomere..

(39) • Telomerase is not present in most cells of multicellular organisms. • Therefore, the DNA of dividing somatic cells and cultured cells does tend to become shorter. • Thus, telomere length may be a limiting factor in the life span of certain tissues and the organism. • Telomerase is present in germ-line cells 生殖細胞, ensuring that zygotes have long telomeres. • Active telomerase is also found in cancerous somatic cells 體細胞. • This overcomes the progressive shortening that would eventually lead to self-destruction of the cancer..

(40) Chromatin Packing in a Eukaryotic Chromosome. 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). • Histones 組蛋白can undergo chemical modifications that result in changes in chromatin organization.

(41) Histone tails. DNA double helix. Amino acids available for chemical modification Nucleosome (end view). 核小體. (a) Histone tails protrude outward from a nucleosome Acetyl groups. Unacetylated histones (side view). DNA. Acetylated histones. (b) Acetylation (乙醯化) of histone tails promotes loose chromatin 染色質 structure that permits transcription 轉錄作用.

(42) Chromatid (700 nm). 30-nm fiber. Loops. Scaffold 300-nm fiber. 30-nm fiber. Chromatin Packing in a Eukaryotic Chromosome. Replicated chromosome (1,400 nm) Looped domains Metaphase 中期 (300-nm fiber) chromosome.

(43) • Chromatin undergoes changes in packing during the cell cycle • At interphase (間期), some chromatin is organized into a 10-nm fiber, but much is compacted into a 30nm fiber, through folding and looping • Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus.

(44) 5 µm. Painting Chromosomes.

(45) • Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis • Loosely packed chromatin is called euchromatin (真染色質) • During interphase a few regions of chromatin, such as centromeres (中節) and telomeres(端粒) are highly condensed into heterochromatin (異染色質) • Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions.

(46) The flow of genetic information. Nuclear envelope. TRANSCRIPTION. RNA PROCESSING. DNA Pre-mRNA. mRNA TRANSCRIPTION. DNA mRNA Ribosome. TRANSLATION. Ribosome. TRANSLATION Polypeptide. Polypeptide. (a) Bacterial cell. (b) Eukaryotic cell.

(47) Reading frame 讀框.

(48) 61 of 64 triplets code. 三聯密碼 for amino acids.

(49) Transcription can be separated into three stages: -Initiation起始 -Elongation延長 -Termination終止.

(50) Complexicity of the transcriptome (轉錄體) (The entire set of transcripts produced in a cell) 200-1000 nucleotides. (Long RNA) 20-200 nucleotides. (Short RNA). (Long noncoding RNA).

(51) Robert Tjian (錢澤南) University of California, Berkeley Howard Hughes Medical Institute. Dynan, W.S. Identified the first transcription specificity protein, Sp1 Transcription factor 轉錄因子 (1983) Cell 32: 669-680. Cell 35: 79-87..

(52) Transcription: the enzymatic production of RNA that complements to its DNA template. Transcription factor 轉錄因子.

(53) The RNA polymerase channels.

(54) General scheme of transcription The transcription bubble 17 bp unwound. E coli RNA polymerase.

(55) Prokaryotic RNA Polymerase RNA polymerase of E. Coli is a multisubunit protein. Subunit α. Number 2. Role Control the frequency of polymerase Initiate transcription from specific promoter. β. 1. β’. 1. Binds DNA template. σ. 1. Recognizes promoter and facilitates initiation (specificity). Forms phosphodiester bond. α2 β β’ ωσ <======> α2 β β’ ω + σ. Holoenzyme. Core enzyme sima factor.

(56) Regulatory gene DNA. Promoter Operator. lacI. lacZ No RNA made. 3′ mRNA. The lac operon in E. coli: regulated synthesis of inducible enzymes. RNA polymerase. 5′. Active repressor. Protein. (a) Lactose absent, repressor active, operon off lac operon. lacI. DNA. lacZ. lacY. lacA. RNA polymerase 3′ mRNA 5′. mRNA 5′. β-Galactosidase. Protein Allolactose (inducer). Inactive repressor. (b) Lactose present, repressor inactive, operon on. Permease. Transacetylase.

(57) trp operon Promoter. Promoter Genes of operon. DNA. trpE. trpR. trpD. trpC. trpB. trpA. C. B. A. Operator Regulatory gene. 3′. RNA polymerase. Start codon. Stop codon. mRNA 5′. mRNA 5′. E Protein. Inactive repressor. D. Polypeptide subunits that make up enzymes for tryptophan synthesis. (a) Tryptophan absent, repressor inactive, operon on. DNA No RNA made mRNA. Protein. Active repressor Tryptophan (corepressor). (b) Tryptophan present, repressor active, operon off. The trp operon in E. coli: regulated synthesis of repressible enzymes.

(58) Promoter. DNA. lacI. lacZ. CAP-binding site. Positive control of. cAMP. Operator RNA polymerase Active binds and transcribes CAP. the lac operon by Inactive CAP Allolactose. catabolite activator protein (CAP). Inactive lac repressor. (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Promoter DNA. lacI. CAP-binding site. lacZ Operator RNA polymerase less likely to bind. Inactive CAP. Inactive lac repressor. (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.

(59) • The diverse functions of RNA range from structural to informational, to catalytic.. Transportation of amino acid into ribosome for protein synthesis.

(60) Classes of Eukaryotic RNA (1) Ribosomal RNA (rRNA) 18 S (small subunit), 28 S (large subunit) 5.8 S (large subunit), 5 S (large subunit) (2) Transfer RNA (tRNA) (3) Messenger RNA (mRNA), microRNA (miRNA) (4) Heterogeneous nuclear RNA (hnRNA) (precursor of mRNA) (5) Small nuclear RNA (snRNA) U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 (6) Small cytoplasmic RNA (scRNA) 7SL RNA (signal recognition particle RNA in protein synthesis).

(61) The Human RNA Polymerases Polymerase. Location. Product. pol I. nucleolus. 18S, 28S,5.8S, rRNA. pol II. nucleoplasm. hnRNA/mRNA, miRNA U1,U2, U4, U5 snRNAs. pol III. nucleoplasm. tRNA, 5S rRNA U6 snRNA, 7SL RNA. Mitochondrial RNA pol. Mitochondria. All mitochondria RNA. t.

(62) An eukaryotic gene and its transcript. Enhancer (distal control elements). Proximal control elements. Transcription start site Exon. DNA Upstream. Intron. Exon. Intron. Downstream Poly-A signal Intron Exon Exon Cleaved 3′ end of primary RNA processing transcript. Promoter. Transcription. Exon. Primary RNA transcript 5′ (pre-mRNA). Poly-A signal Transcription sequence termination region Intron Exon. Intron RNA Coding segment mRNA. G. P. AAA ⋅⋅⋅AAA. P P. 5′ Cap. 5′ UTR. Start Stop codon codon. 3′ UTR Poly-A tail. UTR: untranslated region. 3′.

(63) Promoter. Activators DNA Enhancer. Distal control element. Gene. TATA box General transcription factors. DNAbending protein Group of mediator proteins. A model for the action of RNA polymerase II. enhancers and transcription activators.. RNA polymerase II Transcription initiation complex. RNA synthesis.

(64) Nuclear Architecture and Gene Expression • Loops of chromatin extend from individual chromosomes into specific sites in the nucleus • Loops from different chromosomes may congregate at particular sites, some of which are rich in transcription factors and RNA polymerases • These may be areas specialized for a common function.

(65) Chromosomal interactions in the interphase nucleus. Chromosomes in the interphase nucleus. Chromosome territory. 10 µm. Chromatin loop. Transcription factory.

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(68) RNA splicing is accomplished by a spliceosome. spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs)..

(69) • RNA splicing appears to have several functions. • First, at least some introns contain sequences that control gene activity in some way. • Splicing itself may regulate the passage of mRNA from the nucleus to the cytoplasm. • One clear benefit of split genes is to enable a one gene to encode for more than one polypeptide. • Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons. • Early results of the Human Genome Project indicate that this phenomenon may be common in humans..

(70) • Split genes may also facilitate the evolution of new proteins. • Proteins often have a modular architecture with discrete structural and functional regions called domains. • In many cases, different exons code for different domains of a protein..

(71) Translation.

(72) Structure of tRNA •base pairing between the third base of the codon and anticodon are relaxed (called wobble pairing)..

(73) Atomic resolution structure of the bacterial ribosome. Tome steitz. Ada Yonath. 2009 Nobel prize. Venki Ramakrishnan.

(74) •. Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.. • The P site holds the tRNA carrying the growing polypeptide chain. • The A site carries the tRNA with the next amino acid. • Discharged tRNAs leave the ribosome at the E site..

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(76) • Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits. • First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon. • Initiation factors bring in the large subunit such that the initiator tRNA occupies the P site..

(77) • The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed..

(78) • Termination occurs when one of the three stop codons reaches the A site. • A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site. • This frees the polypeptide and the translation complex disassembles..

(79) • Typically a single mRNA is used to make many copies of a polypeptide simultaneously. • Multiple ribosomes, polyribosomes, may trail along the same mRNA. • A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide..

(80) A summary of genetic flow of an eukaryotic cell..

(81) https://www.youtube.com/w atch?v=7Hk9jct2ozY2021 陳錦翠老師-分生.ppt.

(82) Noncoding RNAs play multiple roles in controlling gene expression • Only a small fraction of DNA codes for proteins, and a very small fraction of the non-proteincoding DNA consists of genes for RNA such as rRNA and tRNA • A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs), such as microRNA (miRNA) • Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration.

(83) • The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) • RNAi is caused by small interfering RNAs (siRNAs) • siRNAs and miRNAs are similar but form from different RNA precursors.

(84) Micro RNAs and Small Interfering RNAs • MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation • It is estimated that expression of at least half of all human genes may be regulated by miRNAs.

(85) Figure 18.14. miRNA miRNAprotein complex 1 The miRNA binds. to a target mRNA.. OR mRNA degraded. Translation blocked. 2 If bases are completely complementary, mRNA is degraded.. If match is less than complete, translation is blocked..

(86) • Small interfering RNAs (siRNAs) are similar to miRNAs in size and function • The blocking of gene expression by siRNAs is called RNA interference (RNAi) • RNAi is used in the laboratory as a means of disabling genes to investigate their function.

(87) Chromatin Remodeling by ncRNAs • Some ncRNAs act to bring about remodeling of chromatin structure • In some yeasts siRNAs re-form heterochromatin at centromeres after chromosome replication.

(88) Condensation of chromatin at the centromere (siRNA-protein complexes) Centromeric DNA. 1 RNA transcripts (red) produced.. RNA polymerase RNA transcript. Sister chromatids (two DNA molecules). 22 Yeast enzyme synthesizes strands. complementary to RNA transcripts.. 3 Double-stranded RNA processed into. siRNAs that associate with proteins.. 4 The siRNA-protein complexes bind. RNA transcripts and become tethered to centromere region.. siRNA-protein complex.

(89) Condensation of chromatin at the centromere. 5 The siRNA-protein complexes recruit. histone-modifying enzymes. Centromeric DNA. Chromatinmodifying enzymes 6 Chromatin condensation is initiated. and heterochromatin is formed.. Heterochromatin at the centromere region.

(90) • Small ncRNAs called piwi-associated RNAs (piRNAs) induce heterochromatin, blocking the expression of parasitic DNA elements in the genome, known as transposons • RNA-based regulation of chromatin structure is likely to play an important role in gene regulation.

(91) The Evolutionary Significance of Small ncRNAs. • Small ncRNAs can regulate gene expression at multiple steps • An increase in the number of miRNAs in a species may have allowed morphological complexity to increase over evolutionary time • siRNAs may have evolved first, followed by miRNAs and later piRNAs.

(92) A program of differential gene expression leads to the different cell types in a multicellular organism • During embryonic development, a fertilized egg gives rise to many different cell types • Cell types are organized successively into tissues, organs, organ systems, and the whole organism • Gene expression orchestrates the developmental programs of animals.

(93) Reprogramming of transcription (Oct4, Sox2, Klf4, and c-Myc) iPS cells Induced pluripotent stem 2012 Noble prize Shinya Yamanaka Kyoto University, Japan John B. Gurdon Gurdon Institute, Cambridge, Embryonic stem cell. United Kingdom 山中伸彌.

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