大学分子生物学经典双语课件.ppt

Chapter 2 Gene and Chromosome,Contents,2.1 Structure of DNA,,Definition：the nucletide residue sequence of the polynucleotide chain； Linkage：3’,5’-phosphodiester bond； Backbone：phosphate + pentose； Direction： 5’ →3’ ；,----DNA double helix,X~ray photograph of DNA with high quality：DNA specimens from different species have the same results（constant width; 3.4nm）； Chargaff rules：the rule of the composition of DNA Physical chemistry studies and acid and alkali titrate studies on DNA base ；,Experimental basis,Three dimensional structure of DNA,10.5 bp /turn,Sugar-P backbone is perpendicular to the planar base pairs,5’,5’,3’,3’,Pitch length,The Watson-Crick B form DNA Deduced by model building,,,,11Å,20Å,Fig 8-15,charged PO4-- on the outside,,,key notes of DNA double helix,Two polynucleotide chains in a DNA double helix; Along the same axis，two chains are wound around each other, resulting in a right-handed double helix; Forms a major groove and a minor groove,The bases lie on the inside，the sugar-phosphate backbone is on the outside; The bases are flat structure, lying in pairs perpendicular to the axis,The diameter of the double helix is 2nm; There is a complete turn every 3.4nm, with 10bp per turn.,2.1.2.1 Stable factors of the double helix,Base-stacking interaction（hydrophobic effect, the major factor）； Hydrogen bond between complementary base pairs； electrovalent bond（between the negative charges carried on the phosphate groups and the positive charges carried on the proteins or metal ions）,2.1.2.2 Conformation polymorphism of the double helix,Alternative double-helical structures of DNA,B-form：relative humidity is 92% A-form：relative devoid of water (under 75%) Z-form：left handed helix H-form：triple helix,1953, Watson & Crick proposed D.S DNA model and found many redundant hydrogen bonding donor and receptors along big grooves. 1957, Felsenfeld proposed T.S DNA concept polyA/polyU polydA/polydT polyd(AG)/polyd(CT) 1983, Mirkin S.M. found plasmid T.S DNA in pH=4.3 solution,1963 Hoogsteen,Triple Helix DNA,1958, Poly(G) X-ray photograph,,Ring structure of hydrogen,,Tetrable helix DNA,Formation condition: polyG, 4(dG),DNA Sculpture at Disneyland,Supercoiling: Supercoiling is the coiling of the DNA axis upon itself。,Linking number(L) Twisting number(T) 缠绕数 Writhing number(W) 扭曲数 L=T+W,Parameters used to express topology properties of DNA:,Positive supercoils,,Negative supercoils,DNA isolated from cell negatively supercoiled by ~5 turns per 100 turns of the helix. Lk / Lk = -0.05,,Positive supercoils,Negative supercoils,Relaxed coils,Topoisomerases,Topoisomerases: exist in cell to regulate the level of supercoiling of DNA molecules Type I topoisomerase: break one strand of the DNA , and change the linking number in steps of ±1 by passing the other strand through the break. Type II topoisomerase: break both strands of the DNA , and change the linking number in steps of ±2 by transferring the other dsDNA through the break.,Type I topoisomerase,Type II topoisomerase,Contents,2.2 Denaturation, renaturation and hybridization,,,2.2.1.1 Denaturation,Definition: a number of physical and chemical factors can lead to the destruction of double-stranded hydrogen-bonded regions of DNA. the double-stranded nucleic acids are converted to single strands.,Denaturation factor,pH（>11.3或<5.0） Chemical denaturation (urea、methanal 甲醛) Thermal denaturation Low ion strength低离子强度,Characters of denatured DNA,biological activity changed (even lost)； viscosity decreased，粘度solubility decreased,溶解度 Hyperchromicity: the absorbance of ssDNA is greater than that dsDNA.增色,melting curve and Tm,Increased temperature can bring about DNA denaturation; Tm （melting temperature): Temperature when 50% DNA denaturation Tm is a characteristic constant of DNA,,94℃,2.2.1.2 Renaturation,Definition：annealing。,D.S DNA,S.S DNA,Denaturation ▲,▼ Renaturation,,,Depends on the collision of complementary S.S. DNA,,,2.2.1.3 renaturation dynamics,DNA 复性过程遵循二级反应动力学 DNA复性过程中单链消失的速度用公式表示： -dC/dt=kC2,,C/C0=1/(1+kC0t),其中，C是单位时间的单链DNA的浓度 C0为开始反应时变性解链的单链DNA浓度， t为复性时间 K是复性速度常数（L/mol·s），k取决于阳离子浓度、温度、pH值、DNA片段大小。,C0t曲线,Definition：the renaturation of regions of complementarity between different nucleic acid strands(DNA or RNA) Characteristic：sensitive、specific,Hybridization of Nucleic Acids,RNA,DNA1,DNA2,DNA,Southern hybridization,Northern hybridization,,,,,,,Juang RH (2004) BCbasics,Probe,Southern blotting Northern blotting dot bloting in situ hybridization Western blotting,Hybridization of Nucleic Acids,Southern blotting,电转印法,Northern Blotting,dot blotting,Denatured DNA or RNA samples were applied directly on a membrane as a dot, then followed by detection by either nucleotide probes.,in situ hybridization,Definition： a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ).,FISH & mFISH,Western blotting,The proteins separated from gel electrophoresis are transferred to a membrane (typically mitrocelulose or PVDF), where they are probed using antibodies specific to the target protein.,Step 1. Antibody Recognition of target protein/antigen,Step 2. Secondary Antibody recognition of primary Ab,Step 3. Color Development,Contents,2.3 Concept of gene,,How to describe genetic material?,Physiological Unit (1864，Herbert Spencer ) Gemmules (1868, Charles Robert Darwin) Germplasm (1883, Weismann) Idioplasm (1884, Karl Wilhelm) Pangen (1889, Hugo de Vries),Mendel’s laws Law of segregation Law of independent assortment,Genetic factor——Gregor Mendel(1865),,Mendel hypothesized genetic factor (alternative forms of genes ) determine heredity.,1909 Wilhelm Johannsen , a Danish botanist, plant physiologist and geneticist. Elemente der exakten Erblichkeitslehre (The Elements of Heredity) . It was in this book introduced the term .,Gene —— Johannsen (1909),Johannsen,gene,Genotype Phenotype,1910 American scientist Morgan suggested that gene was mapped on chromosomes.,Morgan,The gene is viewed as the fundamental unit of mutation, change and function.,1926,2.3.2.1 “One gene, one enzyme” hypothesis,1941, G W Beadle & E L Tatum,Beadle,Tatum,A gene contains the information for producing a specific enzyme.,1951, Yanofsky One gene----one polyeptide chain,,1953, Watson & Crick DNA double helix,Gene is a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome.,2.3.2.2 Theory of Cistron,(1955, S.Benzer),Cistron —— a unit of genetic function defined by a complementation test, which corresponds to an open reading frame.,Benzer,Trans- configuration,Cis-configuration,Complementation test reveals whether two similar mutations are in the same or different genes.,Complementation test between rII－ mutants,Recombination between two mutations in the same gene,Trans-configuration: Unfunctional Functional,,Mutant sites are in different genes.,Mutant sites are in the same gene.,,Cistron —— an alternative forms of genes In a cistron, there are many mutant sites (mutons); In a cistron, there are many crossing over sites (recons). A gene is a cistron, consisting of many mutons and recons.,2.3.2.3 Structural gene and regulatory gene,Operon theory (1961, Jacob F. & Monod J.),2.3.2.4 Splitting gene,1977, Sharp & Roberts,R. J. Roberts P.A. Sharp Nobel Prize 1993,Intron,Exon,Exon: the regions of the DNA that contain coding information, which make up of mature mRNA. Intron: intervening sequences between the coding sequences, they will also be transcribe, but will be removed to yield the mature mRNA.,2.3.2.5 Overlapping gene,赏花归去马如飞，去马如飞酒力微，酒力微醒时已暮，醒时已暮赏花归。 ---苏东坡,Overlapping genes are defined as a pair of adjacent genes whose coding regions are partially overlapping,2.3.2.6 Pseudogene,Pseudogenes are dysfunctional relatives of genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell.,2.3.2.7 Jumping gene,Jumping gene —— a segment of DNA that can become integrated at many different sites along a chromosome (especially a segment of bacterial DNA that can be translocated as a whole),Barbara McClintock,1983 Nobile winner,c) transposable element 是引起玉米糊粉层花斑不稳定 现象的遗传因子,,Ac,,,Ac,,,,,Ds,Ds,CI,SH,2.3.2.8 House keeping gene & Luxury gene,A housekeeping gene is typically a constitutive gene that is required for the maintenance of basic cellular function, and are expressed in all cells of an organism.,A luxury gene codes for specialized cell products and is expressed abundantly. They are tissue-specific or organ-specific, which means they are not expressed in all cells. They are not constantly expressed, only when their function is needed. Examples of luxury genes are plasmids of bacteria and genes coding for heat-shock proteins.,Contents,2.4 Gene cluster and repetitive sequence,,A gene family is a set of several similar genes, formed by duplication of a single original gene, and generally with similar biochemical functions.,Gene family,Gene cluster,A gene cluster is a group of closely related genes that all code for the same function, or variations on the same function.,2.4.1.1 Gene cluster formation,Globin gene 珠蛋白基因,Globin genes are organized in two clusters.,All globin genes have evolved by a series of duplications, transpositions, and mutations from a single ancestral gene.,2.4.1.2 Sequence differentiation,Divergence of DNA sequences depends on evolutionary separation. Each point on the graph represents a pairwise comparison.,Replacement site divergences between pairs of β-globin genes allow the history of the human cluster to be reconstructed. This tree accounts for the separation of classes of globin genes.,Pesudogenes are dead ends of evolution. Pesudogenes(ψ): Some DNA sequences that are related to those of the functional genes, but that cannot be translated into a functional protein.,2.4.1.3 Pesudogene,Many changes have occurred in a β-globin gene since it became a pseudogene.,Pseudogenes could arise by reverse transcription of RNA to give duplex DNAs that become integrated into the genome. 假基因可能在RNA的逆转录时产生，然后被整合到基因组DNA中。,If pseudogenes are evolutionary dead ends, simply an unwanted accompaniment to the rearrangement of functional genes, why are they still present in the genome? Survived in present populations, in the past, any number of other pseudogenes may have been eliminated.,2.4.1.4 Gene cluster rearrangment,Unequal crossing-over rearranges gene clusters.,Gene number can be changed by unequal crossing-over.,Thalassemias result from various deletions in the a-globin gene cluster.,Repetitive sequence —— DNA sequence with high copy numbers.,replication slippage,unequal crossing over,2.4.2.1 Unique sequence,A genome is only one copy or very few copies of DNA sequences . Generally by the coding sequence and interval sequences.,2.4.2.2 moderately repetitive sequence,size：0.1~1kb(less than 10kb) copies：10~10000 including gene cluster and many transposon (sometimes viewed as selfish DNA) functions：do not transcribe, maybe play some role in gene expression,2.4.2.3 highly repetitive sequence,typically <100 bp, thousands of copies, often organized as long tandem repeats； A fraction of this sort is called satellite DNA. mainly near the centromeres (in heterochromatin)； maybe has some structural function in the chromosome. This function could be connected with the process of chromosome segregation；,Satellite DNA,Contents,2.5 Chromosome and nucleosome,,2.5.1.1 prokaryotic chromosome,Procaryotic cell,E. coli,Tobacco Mosaic virus,2.5.1.2 Eukaryotic chromosome,Structure of the nucleus,Animal cell,The eukaryotic cell structure is more complex than prokaryotic；,Component of eukaryotic chromosome: DNA, RNA, Histone, Nonhistones,Chromatin 染色质,,Euchromatin 常染色质,,Heterochromatin 异染色质,Centromere 着丝粒,Telomere 端粒,A telomere is a region of repetitive nucleotide sequences at the end of a chromosome, which protects the end of the chromosome from deterioration（降解） or from fusion（融合） with neighboring chromosomes.,Repeat motif: 5’-T1-4-A0-1-G1-8-3’,Electron micrograph of Drosophila melanogaster chromatin after swelling reveals the presence of nucleosomes as "beads on a string".,2.5.2.1 Subunit of nucleosome,A nucleosome contains ~200 bp of DNA, associated with a histone octamer that consists of two copies each of H2A, H2B, H3, and H4.,2.5.2.2 histone octamer,2.5.2.3 pathway of nucleosome,6 nucleosomes per turn,2.5.2.3 Package of Eukaryotic genome,Compaction ratio = 8400,From DNA to Chromosome,Contents,2.6 Genome,,Genome: all DNA sequences in a cell; a complete genetic sequence on one set of chromosomes.,Maximum C-value: the quantity of DNA in the (haploid) genome. Minimum C-value : the minimum genome size required to make organisms.,Genome size is the total amount of DNA contained within one copy of a single genome.,C-value paradox 悖论,C-value paradox refers to the lack of a correlation between genome size (C-value) and genetic complexity. The minimum genome size found in each phylum increases from prokaryotes to mammals. There are wide variations in the genome sizes of organisms within many phyla, such as insects, amphibians（两栖类动物） and flowering plants. Higher eukaryotes have much more DNA than they “actually need”.,E. coli,Human’s genome,Small; A single closed-circular DNA ； Genes are naked； Most sequences encode genetic information； Regulation units of gene expression----operons操纵子； overlapping genes.,2.6.2.1 Properties of prokaryotic genome,Much larger than prokaryotic chromosome genome； Highly organized complex of DNA and protein, known as chromatin, is consisting of nuclesomes； Each chromosome has multiple origins； a larger proportion of eukaryotic genome does not encode useful genetic information— non-coding DNA (tandem、interspersed)； Repetitive DNA； The coding regions of genes consist predominantly of single-copy genes, which transcribed monocistrons（单顺反子）,2.6.2.2 properties of eukaryotic genome,Contents,2.7 Genetic information flow,,Central Dogma,Crick（1958）,Extended Central Dogma,Table of the 3 classes of information transfer suggested by the dogma,The latest genetic information flow,Thank You!,