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1、Molecular BiologyHongyun Yu2010.3.4Lecturer: 俞红云俞红云 Room: 427Tel: 3600937E-Mail: TA: 龚德顺龚德顺 Tel: 138 6672 4424 E-mail: Lecturer: 涂晓明涂晓明Room: 440ATel: 3600757E-Mail: 课程安排：课程安排： 按大纲顺序进行按大纲顺序进行考试：平时作业及课堂测验考试：平时作业及课堂测验 2530 期末考试期末考试 7075。教材：教材： Essentials iGenetics by Peter J. Russell参考书：参考书： 1. Molec
2、ular Biology of the Gene 5thed by Watson et al Pearson publication 2. Molecular Biology by Robert F. Weaver 3. Genetics by Peter J. Russell 4. Gene VIIIMolecular Biology Class Background Survey1.遗传物质应该具有的基本特征是什么？遗传物质应该具有的基本特征是什么？2.双链双链DNA的基本结构特征是什么？的基本结构特征是什么？3.真核生物染色体是由哪几部分组成的？真核生物染色体是由哪几部分组成的？4.什么
3、是等位基因？什么是等位基因？5.什么是隐性突变？什么是隐性突变？ 当我们说一个基因的当我们说一个基因的某一种突变是隐性致死突变应该有什么依某一种突变是隐性致死突变应该有什么依据（表型）？据（表型）？6.什么是蛋白质的结构域？有什么特点？什么是蛋白质的结构域？有什么特点？Introduction Molecular Genetics vs. Molecular BiologyGeneticsUnderstanding the biological properties that are transmitted from parents to offsprings. The Subjects of
4、 Genetics: 1) Phenomenology of heredity 2) How gene control metabolism & development 3) The distribution & behavior of genes in population Molecular BiologyGeneral: Any Molecular events in an living systemSpecific: Any Molecular events related to genetic materials and genetic information transmissio
5、n. Bio-techniques Genetic analysisThe hypothetico-deductive method 1. Make observations: 自然的、实验产生的自然的、实验产生的2. Formulate hypotheses: hypotheses refined on second and subsequent iterations（反复）（反复）.3. Make experimental predictions: 如果做某个实验，应该有什么结果。如果做某个实验，应该有什么结果。4. Test predictions: produce new observ
6、ations new hypotheses (refined) fruit fly eye color, human color blindnessCriterion of suitable organisms for genetic analysis1.Well known genetic history2.Relatively short life cycle: for data collection over generations3. One mating produce large number of offspring: to get much genetic informatio
7、n. Association study 4.The organisms are easy to handle: mouse vs.elephent5. There must be genetic variation among the individual in the population: for the study of traits inheritance生物学研究所选用的模式生物生物学研究所选用的模式生物因为：同类的生物在分子、细胞、组织、生化途径等因为：同类的生物在分子、细胞、组织、生化途径等各方面有很大程度上的共同性，各方面有很大程度上的共同性，所以：科学家们普遍选用一些有代表
8、性的、个体相对所以：科学家们普遍选用一些有代表性的、个体相对简单、易于操作、便于取得研究数据的生物作为研简单、易于操作、便于取得研究数据的生物作为研究材料，进行各种生物学相关研究，并将相关研究究材料，进行各种生物学相关研究，并将相关研究成果推演到更高级、复杂的生物体系这些生物成果推演到更高级、复杂的生物体系这些生物即为模式生物。即为模式生物。 如：拟南芥、小鼠、斑马鱼、如：拟南芥、小鼠、斑马鱼、Ecoli、非洲爪蟾等。、非洲爪蟾等。How is genetic material transmitted lGregor Mendel 1863: breading of pea, study tr
9、ait (phenotype) behavior (transmission) Conclusion: the phenotypic traits are controlled by factors (genes). Segregation patternlWalter Sutton & Theodor Boveri 1902 : The chromosome theory of Heredity, gene segregation patterns parallel with that of chromosome segregation.Mapping of Genetic material
10、Genetic map: the relative location of genes of the chromosome (crossing) Centimorgan (cM): interval in which 1% crossover take placePhysical map:the real location of genes on chromosome (Sequencing) （bp or kb) Genetic map= physical map?In human beings: 1 centimorgan to 1 million bpFrom genotype to p
11、henotypelGenotype: The genetic makeuplPhenotype: All the measurable attributes GenotypePhenotype？the identical twinslPenitrance of genes (表现度表现度): the degree a gene manifestslHow environment figures in this? Make connection between gene and proteinsl1941,George Beadle & Edward Tatum: fungus metaboli
12、c pathway mutations one gene one enzyme hypothesisGenelA gene is a sequence of nucleotide pairs in DNA that codes for a RNA or Protein product Eukaryotes: introns Prokaryotes Viruses : dsDNA ssDNA RNA ?lCan proteins be genetic material? Mad caw disease Form Conversion to Aggregation:Protofibril Form
13、ation of the Prion ProteinPrPCPrPScLow pHM. L. DeMarco and V. Daggett Proc. Natl. Acad. Sci. USA 101, 2293-2298, 2004. MDThe flow of genetic information translation DNA RNA protein transcription/ reverse transcription mRNA tRNA rRNA snRNAreplicationFrom RNA to ProteinlGenetic code: The base pair inf
14、ormation that potentially specifies the amino acid sequence of a proteinlCodon: The specific three-nucleotide sequence of the mRNA that specifies each amino acid.Gene expression is regulatedlObservation: At any one time ,only some of the genes in a genome are active. In multicellular organisms, only
15、 a specific set of genes are active in specific tissue/organ.lHypotheses: achieved by a finely tuned array of gene regulation signalslThe first model of gene regulation: Jacob & Monod, 1961, the lactose metabolismThe localization of genetic materialNucleusEndoplasmic reticulum (ER)Ribosomes Mitochon
16、driachloroplastsCutaway diagram of a generalized prokaryotic cellThe chloroplast Photosynthesis, dsDNA Mitochondrion Energy processing, dsDNA DNA double helixTranscription Wherever a gene exists on a DNA molecule: One strand is the coding strand (or sense strand or non-template strand), The other is
17、 the noncoding strand (also called the antisense strand antisense is a general term for a sequence of DNA or RNA that is complementary to mRNA , anticoding strand, template strand, or transcribed strand). RibosomeExperimental Experimental approach:approach:CloningCloning Cloning vectorsRestriction e
18、nzymesHistorical landmarksl1866 Gregor Mendel: principle of heredityl1903 W.E.Castle: relationship between allele and genotype frequencyl1911 T.H.Morgan: genetic linkage is the result of the genes involved being on the same chromosomel1944:Avery,MacLeod, McCarty: transforming agent is DNA l1950: B.M
19、cClintock: movable genel1953: Watson & Crick: double helical model of DNA The Streptococcus pneumoniaeDNA as genetic materialGriffiths Transformation ExperimentGriffiths Transformation ExperimentMechanism of Griffiths Transformation RNA as Genetic Material你还能提出其它方法来证明这个结论吗？DNA StructuresModels of B-
20、DNA StructureModels of different dsDNA structurelA B Z A B Z这三种构象在天然条件下都存在吗？DNA can bendlB-conformation is flexiblelSpecific sequences can introduce bend CAAAAAT Protein binding can induce bending Recombination event Restriction enzyme binding Transcription factorsDNA bends upon CAP bindingDNA Bendi
21、ng 的意义何在？How Should We Know About DNA Bending?DNA Bending Affects Electrophoretic MobilityThe organization of DNAGenome DNA organization and the way they are inherited varies with the organismProkaryoteBacterial & dsDNA virus chromosomes: One circular double stranded supercoiled moleculePrediction:
22、form one band after centrifugationExperiment: two bands Explanation: relaxed and supercoiled have different density1,100um(4x103kb), there are histone-like proteins involved in packingSupercoil of circular ds DNA Electron micrographs of bacteriophage plasmid DNA Nucleoid region Nucleoid region relea
23、sed from a lysed released from a lysed E.coli cellE.coli cellBacterial chromosomeThe looped domainsT4 phageCircularly permuted DNA moleculesTerminal redundancy and circular permutationProducing circularly permuted and terminally redundant T4 chromosomeT-even phage chromosomeCircularly permuted: circ
24、ular dsDNA, but the chromosome might have ends at different points around the circular maps, with the ends overlapping. the phage chromosome itself is linearTerminally redundant: the replication and recombination produce one long concatmaric DNA chain, the phage head packing DNA by headful, each is
25、packing a little more than the genome length.Bacteriophage X174Bacteriophage X174Circular ssDNA 5386nt long:Expect: code for 1795 aa at most, MW=19700dFact: code for 11 proteins, total MW=262000dDiscovery: partial overlapping / within overlapping genes with a different reading frame. Gene Organizati
26、on of Bacteriophage X174phage chromosome structure varies at stages of lytic infection of E.colichromosome structurelLinear dsDNA with complementary “sticky” ends (cos) lThe linear DNA circularize upon infectionlHas lysogenic and lytic life cycleslIn lytic cycle, ter binds to the cos sequence, gener
27、ates linear dsDNA with sticky endsEukaryotic ChromosomesStructural characteristics of eukaryotic chromosomesl2 copies of chromosomes in somatic cellslMore than one chromosomes in haploid cellslLarge amount of associating proteins: Histone and non-HistonelMany orders of packing: lCan be devided into
28、unique-sequence and repetitive-sequence. C-value: cellular DNA content (haploid) C-value paradox: Karyotype: a complete set of all the metaphase chromosomes in a cell Chromatin: a chromosome that consists of a complex of DNA; chromosome proteins; and RNA Euchromatin: less condensed, active Heterochr
29、omatin: highly condensed, inactive Human Male Metaphase Chromosome arranged as a karyotypeChromosome associating proteinsHistones: small basic proteins (+) 25% Lys +Arg, conservative H1: H1 has a central conserved core, the flanking regions are less conservative more than one kind in a cell H2A, H2B
30、, H3, H4: very conservativeA Chromatin contains equal weight of DNA and histone proteinsChromosome associating proteinsNon-histones: All the other associating proteins beside Histone Acidic(-), varies from cell to cell, from time to timeLarge varity: Structural & Transient Transients: Replication an
31、d Transcription regulation related Enzymes and Proteins.Amount: weight by weight: Non-histonesDNA+ histonesNucleosomes A possible A possible nucleosomenucleosome structure structure lThe winding of DNA around Histones into a structurelHistone octamer: 2H2A, 2H2B, 2H3, 2H4lLinker H1 molecule: an H1 b
32、etween nucleosomeslDNA: 180 bpNucleosomes Histone proteins in nucleosome have protruded tails that are important in regulating chromosome activity. Lys at the tail can be modified.H2A: light blue, H2B: gray, H3: yellow, H4: slate Nucleosome Assembly 30-nm Chromatin Fiber6 nucleosomes each turnH1 pro
33、tein is necessary30-nm chromatin fiber organize into looped domains (300nm300nm) Different orders（level） of chromatin packing Unique-sequence and Repetitive-sequence.lUnique-sequence DNA: one to a few copieslModerately Repetitive-sequence DNA: a few to 105 copies lHighly Repetitive sequence DNA: 105
34、-107copies1.Tandemly repeated DNA sequences: 2. clusted together, arranged in a row.lDispersed repeated sequences: randomly distributed in genome.Unique-Sequence DNAOne to a few copies of sequencesMost of the genes that code for proteins belong to this classNot all unique-sequence material contain p
35、rotein coding sequences silent geneTandemly repeated DNALow repetitive-sequence: several to hundreds of copies: Genes for rRNA and tRNA; Histone gene of some species; and multi-gene family(-globin)Highly repetitive sequences: simple、short (1-10bp) 、thousands of copies: telomeres/centromeres/ satelli
36、te DNA, may have unusual %GC content. Centromere sequence: 8% in mouse. 50% in kangroo 5% in humanHighly repetitive satellite DNA Microsatellite DNA: 2-4 bp repeatesMinisatellite DNA: 5- a few tens of bp repeatesDispersed repeated sequences 1) Gene sequences: low copy number (50) histones; transposo
37、ns; multigene family 2) Non-gene sequences: high copy number, up to one million, SINE/ LINE. Non-gene sequencesShort interspersed repeats(SINE): 100-500 bp units (Alu I) Use as markers for genome analysisLong interspersed repeats (LINE): 5000bp or longer units. Transposons? FUNCTION? Unknown. Gene e
38、volution? Centromeres and Telomeres Mostly heterochromaticCentromeres: the sites at which chromosomes attach to the mitotic and meiotic spindles. Sequences are conserved to some extent Telomeres: the ends of the chromosomes All telomeres in a given species share a common sequence Centromeres lRespon
39、sibe for the accurate segregation of the replicated chromosomes to the daughter cells lConsequences of nondisjunction: the zygotes mostly die some lives to produce abnormal offspring trisomy 21Centromeres lCentromere sequences are not randomlThere are proteins facilitating spindle fiber attachment t
40、o the centromere: KinetochoreCentromeres of yeastKinetochore of Yeast CDE: Centromere DNA elementsCBF: Centromere binding factorFunctions of Telomeres 1. Prevent exonuclease from degrading chromosomes of the linear DNA ends2. Prevent chromosome ends fusion3. Facilitate replication at the ends withou
41、t losing DNA material.Required for replication and stability of chromosomeWhat happens when chromosome broke?lA broken chromosome may have a sticky ends, which will cause two sticky ends to fuse-resulting one fused chromosome have two or none centromeres and cause problem in mitosis and meiosis.Telo
42、mere sequencesSimple telomere sequences: Simple tandemly repeated, species-specific, at the very end of the chromosome moleculeTelomere-associated sequences: Repeated but still complex sequences, extending thousands of base pairs in from telomere end.Telomere sequences of yeastHow should this sticky end be protected?T-loop at the end of chromosomeElectron micrograph of a T loop at the end of a chromosome isolated from a mouse hepatocyte. The bar at the bottomof the micrograph represents a length of 5,000 bp5,000 bp
