molecular biology fourth editionmcgraw hill …

1、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 bl

2、ood 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 experim

3、ents 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-kille

4、d virulent bacteria could transform avirulent bacteria into virulent bacteria3Outline of Griffiths Transformation Experiments4DNA: The Transforming MaterialIn 1944 Avery, Macleod and McCarty used a transformation test similar to Griffiths procedure taking care to define the chemical nature of the tr

5、ansforming 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:Ultracentrifugatio

6、nUsed 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 fo

7、r 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 propo

8、rtionsIn 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 Chases Experiment8SummaryThe classic molecular biology experiments performed by Griffith, Avery, MacLeod, Mccarty, Hershey

9、 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 sugar10Nucleosi

10、des 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 grou

11、p (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 bondNucleot

12、ides 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 c

13、alled 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 Franklins x-ray diffraction data suggested t

14、hat DNA had a helical shapeThe data also indicated a regular, repeating structureChargaffs 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 He

15、lixStructure 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 backb

16、ones 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

17、 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 RN

18、A 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 h

19、umidity (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

20、 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, ca

21、lled 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 Meltin

22、gWith 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 denatured is the melting temperature or TmGC content of DNA has a significant effect on Tm with high

23、er 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 o

24、f 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 streng

25、th, 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 w

26、ithin 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

27、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 per helical turn of 10.4 base pairsDNA can be measured by electron microsc

28、opy 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 DN

29、A 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?Cant determine from DNA size aloneFactors inc

30、lude: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 avera

31、ge 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 bp4200 proteins Phage l (in