Bài giảng Molecular Biology - Chapter 2 The Molecular Nature of Genes

Molecular BiologyFifth EditionChapter 2The Molecular Nature of GenesLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.1The Nature of Genetic MaterialHistorical BackgroundMiescher isolated nuclei from pus (white blood cells) in 1869Found a novel phosphorus-bearing substance = nucleinNuclein is mostly chromatin, a complex of DNA and chromosomal proteinsEnd of 19th century – DNA and RNA separated from proteinsLevene, Jacobs, et al. characterized basic composition of DNA and RNA2Molecular Foundation: Early experiments that explored the question: What is the genetic material?Key experiments performed by Frederick Griffith in 1928Observed change in Streptococcus pneumoniae — from avirulent (R) rough colonies, bacteria without capsules, to virulent (S) smooth colonies, bacteria that had capsulesResult: Heat-killed virulent bacteria could transform avirulent bacteria into virulent bacteria3Outline of Griffith’s Transformation Experiments4DNA: The Transforming MaterialIn 1944 Avery, Macleod and McCarty used a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substanceTechniques used excluded both protein and RNA as the chemical agent of transformationExclusion of DNA verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent5Analytical ToolsPhysical-chemical analysis has often used:UltracentrifugationUsed to estimate size of materialElectrophoresisIndicated high charge-to-mass ratioUltraviolet Absorption SpectrophotometryAbsorbance of UV light matched that of DNAElementary Chemical AnalysisNitrogen-to-phosphorus ratio of 1.67, expected for DNA but lower than expected for protein6Confirmation for DNA as the genetic materialIn the 1940s geneticists doubted the use of DNA as the genetic material as it appeared to be monotonous repeats of 4 basesBy 1953 Watson & Crick published the double-helical model of DNA structure and Chargaff demonstrated that the 4 bases were not present in equal proportionsIn 1952 Hershey and Chase demonstrated that bacteriophage infection comes from DNA, adding more evidence to support that DNA is the genetic material7Outline of Hershey and Chase’s Experiment8SummaryThe classic molecular biology experiments performed by Griffith, Avery, MacLeod, Mccarty, Hershey and Chase combined revealed that DNA is the genetic element9The Chemical Nature of PolynucleotidesBiochemists determined the components of nucleotides during the 1940sThe component parts of DNANitrogenous bases: Adenine (A)Cytosine (C)Guanine (G)Thymine (T)Phosphoric acidDeoxyribose sugar10Nucleosides and DeoxyriboseRNA component partsNitrogenous basesLike DNA except Uracil (U) replaces ThyminePhosphoric acidRibose sugarBases use ordinary numbersCarbons in sugars are noted as primed numbersNucleotides contain phosphoric acidNucleosides lack the phosphoric acidDeoxyribose lacks a hydroxyl group (OH) at the 2-position11Purines and PyrimidinesAdenine and guanine are related structurally to the parent molecule purineCytosine, thymine and uracil resemble the parent molecule pyrimidine12DNA LinkageNucleotides are nucleosides with a phosphate group attached through a phosphodiester bondNucleotides may contain one, two, or even three phosphate groups linked in a chain13A TrinucleotideThe example trinucleotide has polarityThe top of molecule has a free 5’-phosphate group = 5’ endThe bottom has a free 3’-hydroxyl group = 3’ end14SummaryDNA and RNA are chain-like molecules composed of subunits called nucleotidesNucleotides contain a base linked to the 1’-position of a sugar and a phosphate groupThe phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’-hydroxyl groups by phosphodiester bonds15DNA StructureThe Double HelixRosalind Franklin’s x-ray diffraction data suggested that DNA had a helical shapeThe data also indicated a regular, repeating structureChargaff’s data revealed that the content of purines was always roughly equal to pyrimidinesWatson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned on the interior16DNA HelixStructure compared to a twisted ladderCurving sides of the ladder represent the sugar-phosphate backboneLadder rungs are the base pairsThere are about 10 base pairs per turnArrows indicate that the two strands are antiparallel17SummaryThe DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the insideThe bases pair in a specific way:Adenine (A) with thymine (T)Guanine (G) with cytosine (C)18Genes Made of RNAViruses are a package of genesNo metabolic activity of their ownWhen a virus infects a host cell, the cellular machinery is diverted and begins to make viral proteinsViral genes are replicated and used for the production of viral protein that assemble into virus particlesViruses contain nucleic acid, some viruses use DNA genes, but some viruses have RNA genes, either double- or single-stranded19Physical Chemistry of Nucleic AcidsDNA and RNA molecules can appear in several different structural variantsChanges in relative humidity will cause variation in DNA molecular structureThe twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA also exists and was identified in 197920A Variety of DNA StructuresHigh humidity (92%) DNA is called the B-formReduce relative humidity to 75% and DNA takes on the A-formPlane of base pairs in A-form is no longer perpendicular to the helical axisThe A-form is seen when one strand of DNA is hybridized with one strand of RNA strand When wound in a left-handed helix, DNA is found in the Z-form To date at least one gene requires Z-DNA for activation21SummaryIn the cell, DNA may exist in the common B form, with horizontal base pairsA very small fraction of the DNA may assume a left-handed helical form called the Z-form An RNA-DNA hybrid assumes a third helical shape, called the A-form, with base pairs tilted away from the horizontal22Variation in DNA between OrganismsRatios of G to C and A to T are fixed in any specific organismThe total percentage of G + C varies over a range of 22 to 73%These reflect differences in physical properties23DNA Denaturation or MeltingWith heating, noncovalent forces holding DNA strands together weaken and breakWhen the forces break, the two strands come apart in denaturation or meltingThe temperature at which the DNA strands are ½ denatured is the melting temperature or TmGC content of DNA has a significant effect on Tm with higher GC content yielding a higher Tm24DNA DenaturationIn addition to heat, DNA can be denatured by:Organic solventsHigh pHLow salt concentrationGC content also affects DNA densityDirect, linear relationshipDue to larger molar volume of A-T base pairs compared to G-C base pairs25SummaryThe GC content of a natural DNA can vary from less than 25% to almost 75%The GC content has a strong effect on the physical properties of the DNA, each of which increase linearly with GC contentThe melting temperature, the temperature at which the two strands are half-dissociated or denaturedDensityLow ionic strength, high pH and organic solvents also promote DNA denaturation26DNA RenaturationAfter two DNA strands separate, under proper conditions the strands can come back togetherProcess is called annealing or renaturationThree most important factors:Temperature – best at about 25 C below TmDNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each otherRenaturation Time – as increase time, more annealing will occur27Polynucleotide Chain HybridizationHybridization is a process of putting together a combination of two different nucleic acidsStrands could be 1 DNA and 1 RNAAlso could be 2 DNA with complementary or nearly complementary sequences28DNA SizesDNA size is expressed in 3 different ways:Number of base pairs Molecular weight – 660 is molecular weight of 1 base pairLength – 33.2 Å per helical turn of 10.4 base pairsDNA can be measured by electron microscopy or gel electrophoresis 29DNAs of Various Sizes and ShapesPhage DNA is typically circularSome DNA will be linearSupercoiled DNA coils or wraps around itself like a twisted rubber band30SummaryNatural DNAs come in sizes ranging from several kilobases to thousands of megabasesThe size of a small DNA can be estimated by electron microscopyThis technique can also reveal whether a DNA is circular or linear and whether it is supercoiled31Relationship between DNA Size and Genetic CapacityHow does one know how many genes are in a particular piece of DNA?Can’t determine from DNA size aloneFactors include:How much of the DNA is devoted to genes?What is the space between genes?One can estimate the upper limit of number genes a piece of DNA can hold32DNA Size and Genetic CapacityHow many genes are in a piece of DNA?Start with basic assumptions Genes encode protein (ignoring the RNAs made)The average protein is abut 40,000 D How many amino acids does this represent?Average mass of an amino acid is about 110 DAverage protein – 40,000 / 110 = 364 amino acidsEach amino acid = 3 DNA base pairs364 amino acids requires 1092 base pairs33DNA Genetic CapacityHow large is an average piece of DNA?E. coli chromosome4.6 x 106 bp~4200 proteins Phage l (infects E. coli)4.85 x 104 bp~44 proteinsPhage x174 (one of smallest dsDNAs)5375 bp~5 proteins (squeezes in more by overlapping genes)34DNA Content and the C-Value ParadoxThe C-value is the DNA content per haploid cellOne might expect that more complex organisms need more genes than simple organismsFor the mouse or human compared to yeast this is correctYet the frog has 7 times more genes per cell than humans35C-Value ParadoxThe observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradoxThe most likely explanation for the paradox is that organisms with extraordinarily high C-values simply have a great deal of extra, noncoding DNA36SummaryThere is a rough correlation between DNA content and number of genes in a cell or virusThis correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widelyThe C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms37