ch18.ppt

KeysDescribe types of information that can be obtained by using various techniques.Define magnification and resolution for light microscopy.Describe components of the light and electron microscopes and their functions.Outline different light and electron microscopy techniques emphasizing the information they reveal and their advantages and disadvantages.Outline the factors that cause the formation of different radioisotopes and describe the major methods used to detect radioactivity.Describe cell culture techniques and advantages of working in that system.Discuss various methods used to purify organelles, proteins, and nucleic acids and the traits of these molecules by which the methods effect purification.Outline nucleic acid hybridization techniques and recombinant DNA technology.Discuss polymerase chain reaction and techniques for DNA sequencing.Discuss the production and use of antibodies in research.



IntroductionResearch in cell biology requires complex instrumentation and techniques.Cell and molecular biology is more dependent on the development of new instruments and technologies than any other branch of biology.Understanding the technology helps in understanding the cell.It is important to understand these techniques and the types of information that can be learned by using these techniques. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.1) The Light MicroscopeThe light microscope uses the refraction of light rays to magnify an object.A condenser directs light toward the specimen.The objective lens collects light from the specimen.The ocular lens forms an enlarged, virtual image.

Sectional diagram through a compound light microscope that has both an objective and an ocular lensPaths taken by light rays that form the image of the specimen and those that form the background light of the field. 2013 John Wiley & Sons, Inc. All rights reserved.


ResolutionResolution is the ability to see two nearby points as distinct images.The numerical aperture is a measure of the light-gathering qualities of a lens.The limit of resolution depends on the wavelength of light.Optical flaws, or aberrations, affect resolving power.The Light MicroscopeResolutionMagnification versus resolution: (a) to (b) has increased magnification and resolution, but (b) to (c) only increased magnification Relationship between the stimulation of individual photoreceptors (left) and the resulting scene one would perceive (right). 2013 John Wiley & Sons, Inc. All rights reserved.


VisibilityVisibility deals with factors that allow an object to be observed.It requires that the specimen and the background have different refractive indexes.Translucent specimens are stained with dyes.A bright-field microscope a light that illuminates the specimen is seen as a bright background; it is suited for specimens of high contrast such as stained sections of tissues.The Light MicroscopeVisibilityFeulgen stain: specific for DNA, showing the chromosomes of an onion root tip cell in metaphase of mitosis at the time it was fixed. 2013 John Wiley & Sons, Inc. All rights reserved.


The Light MicroscopeVisibilityPreparation of Specimens for Bright-Field Light MicroscopyA whole mount is an intact object, either living of dead.A section is a very thin slice of an object.To prepare a section, cells are immersed in a chemical called a fixative.The rest of the procedures minimize alteration from the living state. 2013 John Wiley & Sons, Inc. All rights reserved.Feulgen stain: specific for DNA, showing the chromosomes of an onion root tip cell in metaphase of mitosis at the time it was fixed.


Phase-Contrast MicroscopyThe phase-contrast microscope makes highly transparent objects more visible by converting differences in the refractive index of some parts of the specimen into differences in light intensity.Differential interference contrast (DIC) optics gives a three-dimensional quality to the image.The Light MicroscopePhase-contrastA comparison of cells seen with different types of light microscopes: a ciliated protist under bright-field (top), phase-contrast (middle), and differential interference contrast (bottom). 2013 John Wiley & Sons, Inc. All rights reserved.


Fluorescence Microscopy (and Related Fluorescence-Based Techniques)Fluorescence microscopy has made possible advances in live-cell imaging.Fluorochromes are compounds that release visible light upon absorption of UV rays.Fluorochrome stains cause cell components to glow, a phenomenon called fluorescence.Fluorochrome-conjugated antibodies are used to locate specific cellular structures (immunofluorescence).The gene for green fluorescent protein (GFP) from jellyfish can be recombined with genes of interest in model organisms.GFP is expressed with the host gene of interest.GFP is used to follow a gene of interest.The Light MicroscopeFluorescence microscopy 2013 John Wiley & Sons, Inc. All rights reserved.


Fluorescence Microscopy The gene for green fluorescent protein (GFP) from jellyfish can be recombined with genes of interest in model organisms.GFP is expressed with the host gene of interest.GFP is used to follow a gene of interest.The Light MicroscopeFluorescence microscopyUse of GFP variants to follow the dynamic interactions between neurons and target cells in vivo: portion of a transgenic mouse brain with two differently colored fluorescent neurons. 2013 John Wiley & Sons, Inc. All rights reserved.


Fluorescence microscopy A GFP variant is called fluorescence resonance energy transfer (FRET), which uses fluorochromes to measure changes in distance between labeled cellular components.Proteins or individual domains can be fused with different GFP variants.Energy is transferred from one GFP variant to the other GFP variant.The Light MicroscopeFRETFluorescence resonance energy transfer (FRET). This diagram shows an example of the use of FRET technology to follow the change in conformation of a protein (PKG) following cGMP binding. 2013 John Wiley & Sons, Inc. All rights reserved.


Video Microscopy and Image ProcessingVideo microscopy is used to observe living cells.Video cameras offer several advantages for viewing specimens.Special types of video cameras (called charge-coupled device, or CCD cameras) are constructed to be very sensitive to light, which allows them to image specimens at very low illumination.They can detect and amplify very small differences in contrast.Images produced by video cameras can be converted to digital electronic images and processed by a computer.In one technique, the distracting out-of-focus background in a visual field is stored by the computer and then subtracted from the image containing the specimen, greatly increasing the clarity of the image.

The Light MicroscopeVideo microscopy 2013 John Wiley & Sons, Inc. All rights reserved.


Laser Scanning Confocal MicroscopyA laser scanning confocal microscope produces an image of a thin plane located within a much thicker specimen.A laser beam is used to examine planes at different depths in a specimen.The Light MicroscopeLaser scanning confocal microscopyThe light paths in a confocal fluorescence microscope and the focal planeConfocal fluorescence micrographs of three separate optical sections, each 0.3 m thick, of a yeast nucleus stained with two different fluorescently labeled antibodies (DNA in red and a telomere-binding protein in green). 2013 John Wiley & Sons, Inc. All rights reserved.


Super-Resolution Fluorescence MicroscopySTORM (stochastic optical reconstruction microscopy) allows the localization of a single fluorescent molecule within a resolution of

(18.2) Transmission Electron MicroscopeTransmission electron microscopes (TEMs) use electrons instead of light to form images.The limit of resolution is about 10-15 .The resulting images display a 100- to 200-fold increase in resolution over traditional light microscopy.Increased amount of information at the subcellular level.

Comparison of skeletal muscle images from a light (top) and electron (bottom) microscope at a comparable magnification of 4500 times actual size. 2013 John Wiley & Sons, Inc. All rights reserved.


The components of an electron microscope:An electron beam from a tungsten filament accelerated by high voltage, and focused with a magnetic field.A condenser lens is placed between the electron source and the specimen.Differential scattering of electrons by the specimen creates the image.Proportional to the thickness of the specimen.Tissues are stained with heavy metals for contrast.

Transmission Electron MicroscopeA comparison of the lens systems of a light and electron microscope. 2013 John Wiley & Sons, Inc. All rights reserved.


Specimen Preparation for Electron MicroscopySpecimens must be fixed, embedded, and sectioned thinly.Glutaraldehyde and osmium tetroxide are common fixatives.Specimens are dehydrated prior to embedding.Epon or Araldite are common embedding resins.Thin sections cut with glass or diamond knives are collected on grids.Transmission Electron MicroscopySpecimen preparationPreparation of a specimen for observation in the electron microscope. 2013 John Wiley & Sons, Inc. All rights reserved.


Chemicals used may cause artifacts, which may be disproved with other techniques.In negative staining, heavy metal diffuses into spaces between specimen molecules.Shadow casting coats a specimen with metal to produce a three-dimensional effect.

EM: tobacco rattle virus after negative staining with potassium phosphotungstate (top) or shadow casting with chromium (bottom).The procedure used for shadow casting as a means to provide contrast in the electron microscopeTransmission Electron MicroscopyNegative staining & shadow casting 2013 John Wiley & Sons, Inc. All rights reserved.


Transmission Electron MicroscopyFreeze-fracture replication and etchingProcedure for the formation of freeze-fracture replicas and freeze-etching 2013 John Wiley & Sons, Inc. All rights reserved.Freeze-Fracture Replication and Freeze-EtchingIn freeze-fracture replication, frozen tissue is fractured with a knife.A heavy-metal layer is deposited on fractured surface.A cast of the surface is formed with carbon.The metal-carbon replica is viewed in the TEM.In freeze-etching, a layer of ice is evaporated from the surface of the specimen prior to coating it with heavy metal.


Freeze-Fracture Replication and Freeze-EtchingIn freeze-fracture replication, frozen tissue is fractured with a knife.A heavy-metal layer is deposited on fractured surface.A cast of the surface is formed with carbon.The metal-carbon replica is viewed in the TEM.In freeze-etching, a layer of ice is evaporated from the surface of the specimen prior to coating it with heavy metal.Transmission Electron MicroscopyFreeze-fracture replication and etchingReplica of a freeze-fractured onion root cell: nuclear envelope (N.E.) and pores (N.P.), Golgi (G), vacuole (V), and cell wall (C.W.).Deep etching: EM of ciliary axonemes from the protozoan Tetrahymena. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.3) Scanning Electron Atomic Force MicroscopyScanning electron microscopes (SEMs) form images from electrons bounced off the specimen surface.Specimens for SEM are dehydrated by critical-point drying then coated with a layer of carbon, then gold.The image in SEM is indirect.SEM has a wide range of magnification and focus.

SEMs: (left) T4 bacteriophage (X 275,000) and (right) the head of an insect (X 40). 2013 John Wiley & Sons, Inc. All rights reserved.


Scanning Electron Atomic Force MicroscopyAtomic Force Microscopy (AFM)AFM is a high-resolution scanning instrument.AFM provides an image of each individual molecule as it is oriented in the field.A limitation of electron microscopy and X-ray crystallography is that they proved snapshots.AFM can obtain rapid sequential images of a macromolecule so that its activity can be followed over real time.

High-speed atomic force microscopy: Forces between the sample and the AFM tip cause deflections of the tip, which are detected by a laser beam. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.4) The Use of RadioisotopesRadioisotopes can be easily detected and quantified.Properties of radioisotopes:An isotope refers to atoms that differ in the number of neutrons.Isotopes with an unstable combination of protons and neutrons are radioactive.The half-life of a radioisotope measures its instability; half of the radioactive material disintegrates in a given amount of time.Properties of a variety of radioisotopes 2013 John Wiley & Sons, Inc. All rights reserved.


The Use of RadioisotopesRadioisotopes can be easily detected and quantified.Liquid scintillation spectrometryScintillants absorb the energy of an emitted particle and release it in the form of light.Radiation of a tracer in a sample can be detected by measuring light emitted by a scintillant. 2013 John Wiley & Sons, Inc. All rights reserved.Properties of a variety of radioisotopes


Autoradiography is a technique to where a particular isotope is located.A particle emitted from a radioactive atom activates a photographic emulsion.The location of the radioisotope in the specimen is determined by the positions of the overlying silver grains in a photographic emulsion.The Use of RadioisotopesAutoradiography 2013 John Wiley & Sons, Inc. All rights reserved.Preparation of a light microscopic autoradiograph.


(18.5) Cell CultureMost of the study of cells is carried out using cell culture.Cells can be obtained in large quantities.Most culture contain a single type of cell.Many different types of cells can be grown in culture.Cell differentiation can be studied in a cell culture.Cells in a culture require media that includes hormones and growth factors.A primary culture is when cells are obtained directly from the organism.A secondary culture is derived from a previous culture.A cell line refers to cells with genetic modifications that allow them to grow indefinitely.Many types of plant cells can be grown in culture. 2013 John Wiley & Sons, Inc. All rights reserved.


A two-dimensional culture system is when cells are grown on the flat surface of a dish.Labs are moving to three-dimensional cultures in which cells are grown in a 3D matrix consisting of extracellular materials.3D cultures are better suited to study cell-cell interactions.Cell Culture2D vs 3DComparison of the morphology of cells growing in 2D versus 3D cultures: (left) fibroblast in 2D culture is highly flattened with broad lamellipodia, (right) fibroblast in 3D matrix is non-flattened, with a spindle-shaped morphology. White: adhesion sites; blue: fibronectin matrix. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.6) The Fractionation of a Cells Contents by Differential CentrifugationDifferential centrifugation facilitates the isolation of particular organelles in bulk quantity.Prior to centrifugation, cells are broken by mechanic disruption in a buffer solution.The homogenate is subjected to a series of sequential centrifugations.Organelles isolated can be used in a cell-free system to study cellular activitiesPurification of subcellular fractions by density-gradient equilibrium centrifugation: in a continuous sucrose-density gradient different organelles sediment according to density, where they form bands. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.7) Isolation, Purification, and Fractionation of ProteinsProtein purification involves the stepwise removal of contaminants.Purification is measured as an increase in specific activity of a protein.Some identifiable feature of the specific protein must be utilized as an assay to determine the relative amount of the protein.Selective PrecipitationAt low ionic strength, proteins tend to remain in solution.At high ionic strength, protein solubility decreases.Ammonium sulfate is the most commonly used salt for protein precipitation. 2013 John Wiley & Sons, Inc. All rights reserved.


Liquid Column ChromatographyChromatography includes a variety of techniques in which a mixture of dissolved components is fractionated through a porous matrix.Components are fractionated between mobile and immobile phases.The greater the molecules affinity for the matrix, the slower its movement.High performance liquid chromatography (HPLC) has greater resolution due to a tightly packed matrix.Isolation, Purification, and Fractionation of ProteinsLiquid column chromatography 2013 John Wiley & Sons, Inc. All rights reserved.


Ion-exchange chromatography uses ionic charge as a basis for purification.The overall charge of a protein is a summation of all the individual charges of its component amino acids.A pH when the number of positive and negative charges is equal is the isoelectric point.DEAE-cellulose is positively charged and therefore binds negatively charged molecules; it is an anion exchanger.CM-cellulose is negatively charged and acts as a cation exchanger.Isolation, Purification, and Fractionation of ProteinsIon-exchange chromatographyIon-exchange chromatography: separation of two proteins by DEAE-cellulose which binds the negatively charged protein. 2013 John Wiley & Sons, Inc. All rights reserved.


Gel filtration separate proteins by molecular weight.A column is packed with cross-linked polysaccharides of different porosity.Proteins small enough to enter the pores are eluted last.Larger proteins unable to enter the beads remain dissolved in the moving solvent phase.Among those proteins that enter the beads, smaller species are slowed to a greater extent than larger ones.

Isolation, Purification, and Fractionation of ProteinsGel filtration chromatographyGel filtration chromatography: separation of three globular proteins having different molecular mass. Among proteins of similar basic shape, larger molecules are eluted before smaller molecules. 2013 John Wiley & Sons, Inc. All rights reserved.


Affinity chromatography isolates one protein from a mixture using a specific ligand.The technique can achieve near-total purification in a single step.Proteins interact with specific substances: e.g. enzymes with substrates, receptors with ligands, antigens with antibodies.Use column in which the specific interacting molecule is covalently linked to an inert, immobilized material.Isolation, Purification, and Fractionation of ProteinsAffinity chromatographyAffinity chromatography: Schematic of the coated agarose beads to which only a specific protein can combine (top) and steps in the chromatographic procedure (bottom). 2013 John Wiley & Sons, Inc. All rights reserved.


Determining Protein-Protein InteractionsAntibodies establish protein interactions by co-precipitation.The yeast two-hybrid system:A DNA binding domain is linked to the gene for one proteinthe bait protein.An activation domain is linked to genes encoding possible proteins that interact with the bait.A reporter gene (lac Z) is only expressed when the bait and its partner interact.Isolation, Purification, and Fractionation of ProteinsYeast two-hybridUse of the yeast two-hybrid system. 2013 John Wiley & Sons, Inc. All rights reserved.


Polyacrylamide Gel ElectrophoresisElectrophoresis is based on the migration of proteins in an electric field.In polyacrylamide gel electrophoresis (PAGE), proteins are driven through a gel matrix.Movement of proteins depends on molecular size, shape, and charge density.The progress of the gel can be followed using a charged tracking dye.The positions of the proteins can be visualized through autoradiography or Western blot.Isolation, Purification, and Fractionation of ProteinsPolyacrylamide Gel ElectrophoresisPolyacrylamide gel electrophoresis 2013 John Wiley & Sons, Inc. All rights reserved.


Polyacrylamide Gel ElectrophoresisElectrophoresis is based on the migration of proteins in an electric field.SDS-PAGEIt is PAGE carried out in the presence of a charged detergent, sodium dodecyl sulfate (SDS).The repulsion between bound SDS molecules causes the proteins to unfold into a similar shape.Proteins become separated solely on the basis of mass.Isolation, Purification, and Fractionation of ProteinsPolyacrylamide Gel Electrophoresis 2013 John Wiley & Sons, Inc. All rights reserved.Polyacrylamide gel electrophoresis


Two-Dimensional Gel ElectrophoresisIt separates proteins on the basis of both isoelectric focusing and molecular weight.After separation by isoelectric focusing, the gel is removed and subjected to SDS-PAGE.Proteins can then be analyzed mass spectrometry.The technique is ideal for detecting changes in the proteins in a cell under different conditions.Isolation, Purification, and Fractionation of Proteins2D Gel ElectrophoresisTwo-dimensional gel electrophoresis: HeLa cell non-histone chromosomal proteins labeled with [35S]methionine resolving over 1,000 different proteins. 2013 John Wiley & Sons, Inc. All rights reserved.


Protein Measurement and AnalysisThe amount of protein can be determined measuring the amount if light absorbed using a spectrophotometer.Mass spectrometry (MS) measures the mass of molecules, determines chemical formulas and molecular structure, and identifies unknown substances.Protein fragments are converted to ions and separated on the basis of mass and charge.Fragments are compared to large protein databases for identification.

Isolation, Purification, and Fractionation of ProteinsSpectrometryPrinciples of operation of a mass spectrometer 2013 John Wiley & Sons, Inc. All rights reserved.


(18.8) Determining the Structure of Proteins and Multisubunit Complexes X-ray crystallography (or X-ray diffraction) uses protein crystals.Crystals are hit with X-rays, and scattered radiation is collected on a photographic plate.The diffraction pattern provides information about the structure of a protein.The technique is useful in the study of both proteins and nucleic acids.X-ray diffraction analysis. Schematic diagram of the diffraction of X-rays by atoms of one plane of a crystal onto a photographic plate 2013 John Wiley & Sons, Inc. All rights reserved.


Determining the Structure of Proteins and Multisubunit Complexes Information gathered for a protein depends on the resolutionIn myoglobin, 6 resolution shows how polypeptide chain is folded and the location of the heme moiety.At 2 resolution, groups of atoms can be separated from one another.At 1.4 , the positions of individual atoms can be determined.To date, the structures of several hundred proteins have been determined at atomic resolution (1.2 ) and a few as low as 0.66 .Electron density distribution of a small organic molecule (diketopiperazine) calculated at several levels of resolution. 2013 John Wiley & Sons, Inc. All rights reserved.


Determining the Structure of Proteins and Multisubunit Complexes Alternate techniques:In electron cryomicroscopy particles are placed on a grid and rapidly frozen in a hydrated state in liquid nitrogen without being fixed or stained.X-ray crystallography and electron microscopic reconstructions to show how the individual molecules that make up a multisubunit complex interact.Electron crystallography analysis of frozen specimens can also be utilized in the study of membrane proteins in the lipid bilayer.Combining data from electron microscopy and X-ray crystallography: reconstruction of an actin-ADF filament. Actin (red) and ADF molecules (green). 2013 John Wiley & Sons, Inc. All rights reserved.


(18.9) Fractionation of Nucleic AcidsSeparation of DNA by gel electrophoresis.PAGE is used for separation of small DNA and RNA molecules; large ones are separated by agarose.Nucleic acids are separated on the basis of molecular weight.

Gel electrophoresis to separate DNA restriction fragments by sizeDNA fragments in a gel can be revealed by immersing the gel in an ethidium bromide solution and then viewing by ultraviolet light 2013 John Wiley & Sons, Inc. All rights reserved.


UltracentrifugationVelocity Sedimentation: rate at which a molecule moves in response to centrifugal force.Size of organelles and macromolecules expressed in S (Svedberg) units.The S value provides a good measure of relative size.Equilibrium Centrifugation separates nucleic acids based on their buoyant density.Sensitive enough to separate DNA molecules by base composition.

Fractionation of Nucleic AcidsSedimentationSeparation of different-sized DNA molecules by velocity sedimentationSeparation of DNA molecules by equilibrium sedimentation on the basis of differences in base composition 2013 John Wiley & Sons, Inc. All rights reserved.


Fractionation of Nucleic AcidsSedimentationUltracentrifugationVelocity Sedimentation: rate at which a molecule moves in response to centrifugal force.Size of organelles and macromolecules expressed in S (Svedberg) units.The S value provides a good measure of relative size.Equilibrium Centrifugation separates nucleic acids based on their buoyant density.Sensitive enough to separate DNA molecules by base composition.

Fractionating a tubes contents into successive aliquots allows for the absorbance of the solution in each fraction to be measured and plotted. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.10) Nucleic Acid Hybridization Nucleic acid hybridization is based on the ability of two complementary DNA strands to form a double-stranded hybrid.The Southern blot technique is based upon DNA hybridization.The Northern blot technique is based upon RNA-DNA hybridization.Hybridization can be used to determine the degree of similarity between two samples.

Determining the location of specific DNA fragments in a gel by a Southern blot 2013 John Wiley & Sons, Inc. All rights reserved.


(18.11) Chemical Synthesis of DNAChemical synthesis of DNA or RNA supports many other procedures.Hybridization analysis requires single-stranded nucleic acid molecules for use as probes.The chemical reaction linking nucleotides have been automated.A nucleotide is assembled one at a time up to a total of 100 nucleotides.Modifications (biotin and fluorophores) can be incorporated into the molecules.If a double-stranded molecule is needed, it is synthesized as two complementary single strands that can be hybridized together. Longer synthetic molecules are made in segments which are joined together. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.12) Recombinant DNA Technology Recombinant DNA molecules contain DNA sequences derived from more than one source.Restriction endonucleases are enzymes that function in bacteria to destroy viral DNA, restricting the growth of viruses.Are used to dissect genomes into precisely defined fragments for further analysis.Restriction maps are complete diagrams of the fragments that result from digestion of a genome by specific restriction enzymes.

Restriction map construction: small circular genome of the DNA tumor virus polyoma 2013 John Wiley & Sons, Inc. All rights reserved.


Formation of Recombinant DNAsDNA is first cut with restriction enzymes.Recombinant DNAs can be formed in various ways, such as creating sticky ends with restriction enzymes.Two components of a recombinant DNA are linked by DNA ligase.DNA cloning is a technique to produce large quantities of a specific DNA segment.DNA segment to be cloned is first linked to a vector DNA.Bacterial plasmids and viruses are two commonly used vectors.

Recombinant DNA Technology Formation of a recombinant DNA molecule 2013 John Wiley & Sons, Inc. All rights reserved.


Cloning Eukaryotic DNAs in Bacterial PlasmidsPlasmids used for DNA cloning are modified forms of the wild type.Cloning plasmids contain a replication origin.Cloning plasmids usually carry genes for antibiotic resistance.Recombinant plasmids are introduced into bacterial cells by transformation.Plasmid-containing bacteria are selected by treatment with antibiotics.Recombinant DNA TechnologyCloning 2013 John Wiley & Sons, Inc. All rights reserved.Example of DNA cloning with bacterial plasmids


Cloning using plasmidsCells containing various plasmids are grown into separate colonies which can be screened for the presence of a particular DNA sequence.Replica plating produces dishes containing representatives of the same bacterial colonies in the same position in each dish.In situ hybridization uses a labeled DNA probe to locate the colony having the desired DNA fragment.Recombinant DNA TechnologyCloning Replica plating procedure to produce identical copies of the original bacterial plate 2013 John Wiley & Sons, Inc. All rights reserved.


Cloning using plasmidsCells containing various plasmids are grown into separate colonies which can be screened for the presence of a particular DNA sequence.Replica plating produces dishes containing representatives of the same bacterial colonies in the same position in each dish.In situ hybridization uses a labeled DNA probe to locate the colony having the desired DNA fragment.

Recombinant DNA TechnologyCloning 2013 John Wiley & Sons, Inc. All rights reserved.Locating a bacterial colony containing a desired DNA sequence by in situ hybridization


Cloning using plasmidsOnce the colony has been identified, live cells from the colony can be grown into large colonies to amplify the recombinant DNA plasmid.The cells can then be harvested, the DNA extracted and the recombinant plasmid DNA separated from the larger chromosome by equilibrium centrifugation.Recombinant DNA TechnologyCloning Separation of plasmid DNA from that of the main bacterial chromosome by CsCl equilibrium centrifugation 2013 John Wiley & Sons, Inc. All rights reserved.


Cloning Eukaryotic DNAs in Phage GenomesRecombinant DNA molecules are packaged into lambda phage heads.Lambda phage can take inserts up to a size of 25 kb.Sites of phage infection are identified as plaques in a bacterial lawn.DNA fragments are identified by the same techniques as those used for cloning of recombinant plasmids.Recombinant DNA TechnologyCloning Protocol for cloning eukaryotic DNA fragments in lambda phage 2013 John Wiley & Sons, Inc. All rights reserved.


(18.13) Enzymatic Amplification of DNA by PCRPolymerase chain reaction (PCR) is a technique to amplify specific DNA fragments.It uses a very small amount of template.Utilizes a heat-stable DNA polymerase (Taq polymerase) from bacteria living in hot springs.Uses repeated cycles of denaturation, DNA replication, and cooling to double the amount of DNA during each cycle.Uses an automated thermal cycler.Polymerase chain reaction (PCR) 2013 John Wiley & Sons, Inc. All rights reserved.


Applications of PCRAmplifying DNA for cloning or analysisFor criminal investigations, select regions of the genome for amplification that are highly polymorphic so that no two people will have the same results. Fossil analysis from samples millions of years old. Testing for the presence of specific DNA sequencesDetermine whether or not a tissue sample contains a particular virus.Comparing DNA moleculesQuick assays that compare the similarity of two DNA samples such as genomic DNA from bacterial isolates.Quantifying DNA or RNA templatesThe rate of accumulation of product is proportional to the amount of template present in the sample.Enzymatic Amplification of DNA by PCRApplications 2013 John Wiley & Sons, Inc. All rights reserved.


(18.14) DNA SequencingTechniques developed in the 1970s are widely used for sequencing nucleic acids.The Sanger-Coulson dideoxy method became the most widely used:Four samples of identical single-stranded DNA molecules are obtained.DNA of each sample is incubated with a primer, DNA polymerase, four dNTPs, and a low concentration of ddNTPs (dideoxyribonucleoside triphosphates), different one in each sample.DNA fragments of different lengths are synthesized in each sample, with synthesis terminating where ddNTP has been randomly incorporated.

The basic steps in sequencing a small hypothetical fragment by the Sanger-Coulson (dideoxy) technique 2013 John Wiley & Sons, Inc. All rights reserved.


DNA SequencingTechniques developed in the 1970s are widely used for sequencing nucleic acids.The Sanger-Coulson dideoxy method became the most widely used:DNA fragments are separated by gel electrophoresis and the DNA sequence is read from the gel bands.The amino acid sequence of the protein is deduced from the nucleotide sequence.Gel lanes in which fluorescently labeled daughter molecules have been separated. The color of the band is determined by the identity of the dideoxynucleotide at the 3 end of the DNA strandNucleotides in the template strand is interpreted by a computer that reads from the bottom to the top of the gel to generate an electropherogram 2013 John Wiley & Sons, Inc. All rights reserved.


Next-generation sequencing (NGS) is based on polymerase-dependent DNA synthesis but does not depend upon premature termination.NGS does not require separation of strands by electrophoresis.It identifies the individual nucleotides as they are being incorporated by the polymerase in real time.Third-generation (or single molecule) sequencingDoes not require amplified DNA, nor do they involve enzymatic activities.Accomplishes sequencing by pulling each DNA molecule through a tiny hole, or nanopore and identifying each nucleotide one at a time. DNA SequencingNewer techniques 2013 John Wiley & Sons, Inc. All rights reserved.


(18.15) DNA LibrariesDNA libraries are often produced from DNA cloning:Genomic libraries are produced from DNA extracted from nuclei and contain all DNA sequences of the species.DNA fragments for the library can be obtained by cutting genomic DNA with restriction enzymes.Cleaving of genomic DNA is random, which generates overlapping fragments.Overlapping fragments are useful for chromosome walking, to study linked sequences in an extended region of a chromosome.Cloning Larger DNA Fragments in Specialized Cloning VectorsA yeast artificial chromosome (YAC) can accommodate large (up to 1000 kb) DNA inserts.A bacterial artificial chromosome (BAC) accepts DNA inserts of up to 500 kb, and can be quickly grown to large numbers.



DNA LibrariescDNA librariesDNA libraries are often produced from DNA cloning:cDNA libraries are derived from DNA copies of an RNA population.The isolation of genomic fragments allows study of the genome.Coding regions of a gene can be studied using cDNAs, synthesized by reverse transcriptase using mRNA as a template.Allow foreign DNA to be transcribed and translated during the infection process.Synthesizing cDNAs for cloning in a plasmid: the generation of double-stranded DNA from mRNA (left), and cloning the cDNA into a plasmid (right). 2013 John Wiley & Sons, Inc. All rights reserved.


(18.16) DNA Transfer into Eukaryotic Cells and Mammalian EmbryosDNA incorporation into the genome of a non-replicating virus is called transduction.DNA introduced into cultured cells is called transfection.The gene whose role is being investigated after transfection is called a transgene.A direct way to introduce foreign genes into a cell is by microinjection of DNA directly into the cell nucleus.Animals that have been genetically engineered to that their chromosomes have foreign genes are called transgenic animals.

Microinjection of DNA into the nucleus of a recently fertilized mouse eggTransgenic mice: larger mouse (left) has the rat growth hormone gene regulated by the metallothionein promoter 2013 John Wiley & Sons, Inc. All rights reserved.


Transgenic Plants and AnimalsTransgenic organisms allow scientists to determine the effects of overexpression of a particular DNA sequence.Genetic engineering can produce animal models used to study human diseases.The main goal of genetic engineering in plants is to improve the efficiency of both photosynthesis and nitrogen fixation.DNA Transfer into Eukaryotic Cells and Mammalian Embryos 2013 John Wiley & Sons, Inc. All rights reserved.Formation of transgenic plants using the Ti plasmid


(18.17) Determining Eukaryotic Gene Function by Gene EliminationThis process of learning about genotypes by studying mutant phenotypes is known as forward genetics.The process of reverse genetics has recently been developed, which is based on determining phenotype (i.e., function) based on the knowledge of genotypeIn Vitro MutagenesisSite-directed mutagenesis (SDM) allows making small changes in a DNA sequence.SDM is accomplished by synthesizing a DNA containing the desired change and allowing it to hybridize to a single-stranded normal DNA.The polymerase elongates the replicates DNA adding nucleotides complementary to the normal DNA. 2013 John Wiley & Sons, Inc. All rights reserved.


Determining Eukaryotic Gene Function by Gene Elimination: Knockout miceKnockout MiceKnockout mice are obtained by transfecting embryonic stem cells, introducing them into an embryo, and implanting the embryo into a female mouse.Germ cells containing the knockout mutation are heterozygous, which can be used to obtain a homozygous mutant phenotype.Genes can be assessed for their functions.

Formation of knockout mice: step 1 relies on derivation of ES cells from the blastocyst and transfecting those ES cells with a targeting vector 2013 John Wiley & Sons, Inc. All rights reserved.


Determining Eukaryotic Gene Function by Gene Elimination: Knockout miceKnockout MiceKnockout mice are obtained by transfecting embryonic stem cells, introducing them into an embryo, and implanting the embryo into a female mouse.Germ cells containing the knockout mutation are heterozygous, which can be used to obtain a homozygous mutant phenotype.Genes can be assessed for their functions.

Formation of knockout mice: step 2 shows the antibiotic selection process. Once targets have been identified by PCR or Southern blot, ES cells are used for blastocyst injections 2013 John Wiley & Sons, Inc. All rights reserved.


Knockout MiceKnockout mice are obtained by transfecting embryonic stem cells, introducing them into an embryo, and implanting the embryo into a female mouse.Germ cells containing the knockout mutation are heterozygous, which can be used to obtain a homozygous mutant phenotype.Genes can be assessed for their functions.

Formation of knockout mice: step 3 shows the generation of a chimera, derived from both ES cells (generates brown coat color) and the host blastocyst (generates black coat color)Determining Eukaryotic Gene Function by Gene Elimination: Knockout mice 2013 John Wiley & Sons, Inc. All rights reserved.


Knockout MiceKnockout mice are obtained by transfecting embryonic stem cells, introducing them into an embryo, and implanting the embryo into a female mouse.Germ cells containing the knockout mutation are heterozygous, which can be used to obtain a homozygous mutant phenotype.Genes can be assessed for their functions.

Formation of knockout mice: step 4 goes through the breeding strategy to demonstrate germline transmission, and how to make heterozygous and homozygous miceDetermining Eukaryotic Gene Function by Gene Elimination: Knockout mice 2013 John Wiley & Sons, Inc. All rights reserved.


RNA InterferenceSpecific mRNAs can be degraded in vivo by treating with small double-stranded siRNA containing part of the sequence of the target mRNA.Cells treated with RNAi cannot make the protein encoded in the target mRNA.Libraries containing thousands of siRNAs are available for the study of gene function.Determining Eukaryotic Gene Function by Gene Elimination: RNA interferenceDetermining gene function by RNA interference: a gallery of images of cultured Drosophila cells incubated with various double-stranded siRNAs. Cells are screened for the presence of abnormal mitotic spindles by arresting in metaphase. 2013 John Wiley & Sons, Inc. All rights reserved.


RNA InterferenceSpecific mRNAs can be degraded in vivo by treating with small double-stranded siRNA containing part of the sequence of the target mRNA.Cells treated with RNAi cannot make the protein encoded in the target mRNA.Libraries containing thousands of siRNAs are available for the study of gene function.Determining Eukaryotic Gene Function by Gene Elimination: RNA interferenceDetermining gene function by RNA interference: (Left) An untreated, cultured mammalian cell at metaphase of mitosis. (Right) The same type of cell treated by RNAi to knockdown Aurora B kinase, a protein involved in the metaphase spindle checkpoint. 2013 John Wiley & Sons, Inc. All rights reserved.


(18.18) The Use of AntibodiesAntibodies are highly specific proteins produced by lymphoid tissues in response to the presence of foreign materials.Preparation of antibodies:A population of polyclonal antibodies can be obtained by repeated injections of a purified antigen into an animal.The blood of the animal serves as a source of an antiserum.A monoclonal antibody is produced by descendants of a single antibody-producing cell:Antibody-producing cells do not grow and divide in culture.Fusion of a normal antibody-producing lymphocyte and a malignant myeloma cell will create a viable hybridoma cell that can produce large amounts of a monoclonal antibody.



The Use of AntibodiesA monoclonal antibody is produced by descendants of a single antibody-producing cell:Antibody-producing cells do not grow and divide in culture.Fusion of a normal antibody-producing lymphocyte and a malignant myeloma cell will create a viable hybridoma cell that can produce large amounts of a monoclonal antibody.

Formation of monoclonal antibodies 2013 John Wiley & Sons, Inc. All rights reserved.


Antibodies can be conjugated with a fluorescent substance that allows visualization of antigens.In a Western blot, antibodies can be used in conjunction with various types of fractionation procedures to identify a particular protein (antigen) among a mixture of proteins. Immunoprecipitation can be performed to determine protein-protein interaction.Immunohistochemistry/immunocytochemistry can be done to localize proteins within a histological sample or cell.In direct immunofluorescence, antibodies with bound fluorescent molecules bind to antigens and can be visualized with a fluorescence microscope.In indirect immunofluorescence, cells are incubated with unlabeled antibodies, and then with labeled 2 antibody against the 1 antibody.The Use of Antibodies 2013 John Wiley & Sons, Inc. All rights reserved.


Copyright 2013 John Wiley & Sons, Inc.All rights reserved. Reproduction or translation of this work beyond that permitted in section 117 of the 1976 United States Copyright Act without express permission of the copyright owner is unlawful. Request for further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. The purchaser may make back-up copies for his/her own use only and not for distribution or resale. The Publisher assumes no responsibility for errors, omissions, or damages caused by the use of these programs or from the use of the information herein. 2013 John Wiley & Sons, Inc. All rights reserved.


The use of dual-label fluorescence to follow dynamic events within cellular organelles of living cells. The Golgi cisternae of a budding yeast cell are not organized into distinct stacks as in most eukaryotic cells but are dispersed within the cytoplasm. Each of the brightly colored oval structures is an individual cisterna whose color is due to the localization of fluorescently labeled protein molecules. A cisterna that appears green contains a GFP-labeled protein (Vrg4) that is involved in early Golgi activities, whereas a cisterna that appears red contains a DsRed-labeled protein (Sec7) that is involved in late Golgi activities. This series of micrographs reveals the protein composition of individual cisternae over a period of about 13 minutes (elapsed time is indicated in the lower left corner). The white arrowhead and arrow point out two of these cisternae over time. These two cisternae are shown imaged by themselves in the lower rows of micrographs. It can be seen from this series that the protein composition of an individual cisterna changes over time from one containing early Golgi proteins (green) to one containing late Golgi proteins (red).*Figure 18.1Sectional diagram through a compound light microscope; that is, a microscope that has both an objective and an ocular lens.

Figure 18.2The paths taken by light rays that form the image of the specimen and those that form the background light of the field. Light rays from the specimen are brought to focus on the retina, whereas background rays are out of focus, producing a diffuse bright field. As discussed later in the text, the resolving power of an objective lens is proportional to the sine of the angle. Lenses with greater resolving power have shorter focal lengths, which means that the specimen is situated closer to the objective lens when it is brought into focus.

Figure 18.3a-cMagnification versus resolution. The transition from (A) to (B) provides the observer with increased magnification and resolution, whereas the transition from (B) to (C) provides only increased magnification (empty magnification). In fact, the quality of the image actually deteriorates as empty magnification increases.

Figure 18.4The resolving power of the eye. A highly schematic illustration of the relationship between the stimulation of individual photoreceptors (left) and the resulting scene one would perceive (right). The diagram illustrates the value of having the image fall over a sufficiently large area of the retina.

Figure 18.5The Feulgen stain. This staining procedure is specific for DNA as indicated by the localization of the dye to the chromosomes of this onion root tip cell that was in metaphase of mitosis at the time it was fixed.

Figure 18.5The Feulgen stain. This staining procedure is specific for DNA as indicated by the localization of the dye to the chromosomes of this onion root tip cell that was in metaphase of mitosis at the time it was fixed.

Figure 18.6a-cA comparison of cells seen with different types of light microscopes. Light micrographs of a ciliated protist as observed under bright-field (A), phase-contrast (B), and differential interference contrast (DIC) (or Nomarski) optics (C). The organism is barely visible under bright-field illumination but clearly seen under phase-contrast and DIC microscopy.

Figure 18.7a, bUse of GFP variants to follow the dynamic interactions between neurons and their target cells in vivo. (A) A portion of the brain of a mouse with two differently colored, fluorescent neurons. These mice are generated by mating transgenic animals whose neurons are labeled with one or the other fluorescent protein. (B) Fluorescence micrograph of portions of two neurons, one labeled with YFP and the other with CFP. The arrows indicate two different neuromuscular junctions on two different muscle fibers in which the YFP-labeled axonal branch has outcompeted the CFP-labeled branch. The third junction is innervated by the CFP-labeled axon in the absence of competition.

Figure 18.8Fluorescence resonance energy transfer (FRET). This schematic diagram shows an example of the use of FRET technology to follow the change in conformation of a protein (PKG) following cGMP binding. The two small, barrel-shaped fluorescent proteinsenhanced CFP (ECFP) and enhanced YFP (EYFP)are shown in their fluorescent color. In the absence of cGMP, excitation of ECFP with light of 440 nm leads to emission of light of 480 nm from the fluorescent protein. Following cGMP binding, a conformational change in PKG brings the two fluorescent proteins into close enough proximity for FRET to occur. As a result, excitation of the ECFP donor with light of 440 nm leads to energy transfer to the EYFP acceptor and emission of light of 535 nm from the acceptor. The enhanced versions of these proteins have greater fluorescence intensity and tend to be more stable than the original protein molecule.

Figure 18.9a, bLaser scanning confocal fluorescence microscopy. (A) The light paths in a confocal fluorescence microscope. Light of short (blue) wavelength is emitted by a laser source, passes through a tiny aperture, and is reflected by a dichroic mirror (a type of mirror that reflects certain wavelengths and transmits others) into an objective lens and focused onto a spot in the plane of the specimen. Fluorophores in the specimen absorb the incident light and emit light of longer wavelength, which is able to pass through the dichroic mirror and come to focus in a plane that contains a pinhole aperture. The light then passes into a photomultiplier tube that amplifies the signal and is transmitted to a computer which forms a processed, digitized image. Any light rays that are emitted from above or below the optical plane in the specimen are prevented from passing through the pinhole aperture and thus do not contribute to formation of the image. This diagram shows the illumination of a single spot in the specimen. Different sites within this specimen plane are illuminated by means of a laser scanning process. The diameter of the pinhole aperture is adjustable. The smaller the aperture, the thinner the optical section and the greater the resolution, but the less intense the signal. (B) Confocal fluorescence micrographs of three separate optical sections, each 0.3 m thick, of a yeast nucleus stained with two different fluorescently labeled antibodies. The red fluorescent antibody has stained the DNA within the nucleus, and the green fluorescent antibody has stained a telomere-binding protein that is localized at the periphery of the nucleus.

Figure 18.10a-cBreaking the light microscopes limit of resolution. (A) A conventional fluorescence micrograph of a portion of a cultured mammalian cell with microtubules labeled in green and clathrin-coated pits in red. At this high level of magnification, the image appears pixelated. Moreover, the overlap between the two fluorescent labels produces an orange color, which suggests that the two structures are interconnected. (B) A STORM superresolution micrograph of a similar field showing the microtubules and clathrin-coated pits clearly resolved and spatially separated from one another. (C) A magnified view of a portion of part B.

Figure 18.11a, bA comparison between the information contained in images taken by a light and electron microscope at a comparable magnification of 4500 times actual size. (A) A photo of skeletal muscle tissue that had been embedded in plastic, sectioned at 1 m, and photographed with a light microscope under an oil immersion objective lens. (B) An adjacent section to that used for part A that was cut at 0.025 m and examined under the electron microscope at comparable magnification to that in A. The resulting image displays a one- to two-hundred-fold increase in resolution. Note the difference in the details of the muscle myofibrils, mitochondria, and the capillary containing a red blood cell. Whereas the light microscope cannot provide any additional information to that of A, the electron microscope can provide much more information, producing images, for example, of the structure of the individual membranes within a small portion of one of the mitochondria.

Figure 18.12A comparison of the lens systems of a light and electron microscope.

Figure 18.13Preparation of a specimen for observation in the electron microscope.

Figure 18.14a, bExamples of negatively stained and metal-shadowed specimens. Electron micrographs of a tobacco rattle virus after negative staining with potassium phosphotungstate (A) or shadow casting with chromium (B).

Figure 18.15The procedure used for shadow casting as a means to provide contrast in the electron microscope. This procedure is often used to visualize small particles, such as the viruses shown in the previous figure. DNA and RNA molecules are often made visible by a modification of this procedure known as rotary shadowing in which the metal is evaporated at a very low angle while the specimen is rotated.

Figure 18.16Procedure for the formation of freeze-fracture replicas as described in the text. Freeze etching is an optional step in which a thin layer of covering ice is evaporated to reveal additional information about the structure of the fractured specimen.

Figure 18.17Replica of a freeze-fractured onion root cell showing the nuclear envelope (N.E.) with its pores (N.P.), the Golgi complex (G), a cytoplasmic vacuole (V), and the cell wall (C.W.).

Figure 18.18Deep etching. Electron micrograph of ciliary axonemes from the protozoan Tetrahymena. The axonemes were fixed, frozen, and fractured, and the frozen water at the surface of the fractured block was evaporated away, leaving a portion of the axonemes standing out in relief, as visualized in this metal replica. The arrow indicates a distinct row of outer dynein arms.

Figure 18.19a, bScanning electron microscopy. Scanning electron micrographs of (A) a T4 bacteriophage (X 275,000) and (B) the head of an insect (X 40).

Figure 18.20High-speed atomic force microscopy. The basic elements of an HS-AFM are shown in this schematic drawing. The sample is mounted on a component (the peizo actuator) that is attached to the microscope stage (not shown). Signals from the actuator cause the cantilever to oscillate up and down so that the AFM tip intermittently contacts the sample as it scans over the samples surface. Forces that develop between the sample and the AFM tip cause deflections of the tip, which are detected by a laser beam that is reflected off of the back of the AFM tip. Movements in the position of the laser beam, which reflect topographical differences along the sample surface, are recognized by a position detector and this information is used to construct an image of the specimen. The AFM-based movie of the movement of a myosin V molecule shown in Chapter 9 was filmed at approximately 7 frames per second.Figure 18.21a, bLight microscopic autoradiography. (A) Preparing a light microscopic autoradiograph. (B) Light microscopic autoradiograph of a polytene chromosome from the insect Chironomus, showing extensive incorporation of [3H]uridine into RNA in the puffed regions of the chromosome. Autoradiographs of this type confirmed that the chromosome puffs are sites of transcription. This micrograph shows the same chromosome as seen in the scanning electron microscope.

Figure 18.22a, bA comparison of the morphology of cells growing in 2D versus 3D cultures. (A) A human fibroblast growing on a flat, fibronectin-coated substratum in 2D culture assumes a highly flattened shape with broad lamellipodia. Integrin-containing adhesions appear white. (B) The same type of cell growing in a 3D matrix assumes a non-flattened, spindle-shaped morphology. The extracellular fibronectin matrix appears in blue. Bar equals 10 m.

Figure 18.23Purification of subcellular fractions by density-gradient equilibrium centrifugation. In this particular example, the medium is composed of a continuous sucrose-density gradient, and the different organelles sediment until they reach a place in the tube equal to their own density, where they form bands.

Figure 18.24Ion-exchange chromatography. The separation of two proteins by DEAE-cellulose. In this case, a positively charged ion-exchange resin is used to bind the negatively charged protein.

Figure 18.25Gel filtration chromatography. The separation of three globular proteins having different molecular mass, as described in the text. Among proteins of similar basic shape, larger molecules are eluted before smaller molecules.

Figure 18.26a, bAffinity chromatography. (A) Schematic representation of the coated agarose beads to which only a specific protein can combine. (B) Steps in the chromatographic procedure.

Figure 18.27a-eUse of the yeast two-hybrid system. This test for protein protein interaction depends on a cell being able to put together two parts of a transcription factor. (A) The two parts of the transcription factor the DNA-binding domain and the activation domainare seen here as the transcription factor binds to the promoter of a gene ( lacZ) encoding -galactosidase. (B) In this case, a yeast cell has synthesized the DNA-binding domain of the transcription factor linked to a known bait protein X. This complex cannot activate transcription. (C) In this case, a yeast cell has synthesized the activation domain of the transcription factor linked to an unknown fish protein Y. This complex cannot activate transcription. (D) In this case, a yeast cell has synthesized both X and Y protein constructs, which reconstitutes a complete transcription factor, allowing lacZ expression, which is readily detected. (E) If the second DNA had encoded a protein, for example, Z, that could not bind to X, no expression of the reporter gene would have been detected.

Figure 18.28Polyacrylamide gel electrophoresis. The protein samples are typically dissolved in a sucrose solution whose density prevents the sample from mixing with the buffer and then loaded into the wells with a fine pipette as shown in step 1. In step 2, a direct current is applied across the gel, which causes the proteins to move into the polyacrylamide along parallel lanes. When carried out in the detergent SDS, which is usually the case, the proteins move as bands at rates that are inversely proportional to their molecular mass. Once electrophoresis is completed, the gel is removed from the glass frame and stained in a tray (step 3).

Figure 18.28Polyacrylamide gel electrophoresis. The protein samples are typically dissolved in a sucrose solution whose density prevents the sample from mixing with the buffer and then loaded into the wells with a fine pipette as shown in step 1. In step 2, a direct current is applied across the gel, which causes the proteins to move into the polyacrylamide along parallel lanes. When carried out in the detergent SDS, which is usually the case, the proteins move as bands at rates that are inversely proportional to their molecular mass. Once electrophoresis is completed, the gel is removed from the glass frame and stained in a tray (step 3).

Figure 18.29Two-dimensional gel electrophoresis. A two-dimensional polyacrylamide gel of HeLa cell nonhistone chromosomal proteins labeled with [35S]methionine. Over a thousand different proteins can be resolved by this technique.

Figure 18.30Principles of operation of a mass spectrometer.

Figure 18.31X-ray diffraction analysis. Schematic diagram of the diffraction of X-rays by atoms of one plane of a crystal onto a photographic plate. The ordered array of the atoms within the crystal produces a repeating series of overlapping circular waves that spread out and intersect the film. As with diffraction of visible light, the waves form an interference pattern, reinforcing each other at some points on the film and canceling one another at other points.

Figure 18.32a-dElectron density distribution of a small organic molecule (diketopiperazine) calculated at several levels of resolution. At the lowest resolution (A), only the ring nature of the molecule can be distinguished, whereas at the highest resolution (D), the electron density around each atom (indicated by the circular contour lines) is revealed.

Figure 18.33Combining data from electron microscopy and X-ray crystallography provides information on proteinprotein interactions and the structure of multisubunit complexes. The electron microscopic reconstruction of an actin-ADF filament is shown in grey. The high-resolution X-ray crystal structures of individual actin monomers (red) and ADF molecules (green) have been fitted into the lower resolution EM structure.

Figure 18.34a, bSeparation of DNA restriction fragments by gel electrophoresis. (A) DNA is incubated with a restriction enzyme, which cuts it into fragments. The mixture of fragments is introduced into a slot, or well, in a slab of agarose and an electric current is applied. The negatively charged DNA molecules migrate toward the positive electrode and separate by size. (B) All of the DNA fragments that are present in a gel can be revealed by immersing the gel in a solution of ethidium bromide and then viewing the gel under an ultraviolet light.

Figure 18.35a-cTechniques of nucleic acid sedimentation. (A) Separation of different-sized DNA molecules by velocity sedimentation. The sucrose density gradient is formed within the tube (step 1) by allowing a sucrose solution of increasing concentration to drain along the wall of the tube. Once the gradient is formed, the sample is carefully layered over the top of the gradient (steps 2 and 3), and the tube is subjected to centrifugation (e.g., 50,000 rpm for 5 hours) as illustrated in step 4. The DNA molecules are separated on the basis of their size (step 5). (B) Separation of DNA molecules by equilibrium sedimentation on the basis of differences in base composition. The DNA sample is mixed with the CsCl solution (step 1) and subjected to extended centrifugation (e.g., 50,000 rpm for 72 hours). The CsCl gradient forms during the centrifugation (step 2), and the DNA molecules band in regions of equivalent density (step 3). (C) The tube from the experiment of B is punctured and the contents are allowed to drip into successive tubes, thereby fractionating the tubes contents. The absorbance of the solution in each fraction is measured and plotted as shown.

Figure 18.35a-cTechniques of nucleic acid sedimentation. (A) Separation of different-sized DNA molecules by velocity sedimentation. The sucrose density gradient is formed within the tube (step 1) by allowing a sucrose solution of increasing concentration to drain along the wall of the tube. Once the gradient is formed, the sample is carefully layered over the top of the gradient (steps 2 and 3), and the tube is subjected to centrifugation (e.g., 50,000 rpm for 5 hours) as illustrated in step 4. The DNA molecules are separated on the basis of their size (step 5). (B) Separation of DNA molecules by equilibrium sedimentation on the basis of differences in base composition. The DNA sample is mixed with the CsCl solution (step 1) and subjected to extended centrifugation (e.g., 50,000 rpm for 72 hours). The CsCl gradient forms during the centrifugation (step 2), and the DNA molecules band in regions of equivalent density (step 3). (C) The tube from the experiment of B is punctured and the contents are allowed to drip into successive tubes, thereby fractionating the tubes contents. The absorbance of the solution in each fraction is measured and plotted as shown.

Figure 18.36Determining the location of specific DNA fragments in a gel by a Southern blot. As described in the figure, the fractionated DNA fragments are washed out of the gel and trapped onto a nitrocellulose membrane, which is incubated with radioactively labeled DNA (or RNA) probes. The location of the hybridized fragments is determined autoradiographically. During the blotting procedure, capillary action draws the buffer upward into the paper towels. As the buffer moves through the electrophoretic gel, it dissolves the DNA fragments and transfers them to the surface of the adjacent membrane.

Figure 18.37a, bThe construction of a restriction map of the small circular genome of the DNA tumor virus polyoma. (A) Autoradiographs of 32P-labeled DNA fragments that have been subjected to gel electrophoresis. The gel on the left shows the pattern of DNA fragments obtained after a complete digestion of the polyoma genome with the enzyme HpaII. To determine how these eight fragments are pieced together to make up the intact genome, it is necessary to treat the DNA in such a way that overlapping fragments are generated. Overlapping fragments can be produced by treating the intact genome with a second enzyme that cleaves the molecule at different sites, or by treating the genome with the same enzyme under conditions where the DNA is not fully digested as it was in the left gel. The two gels on the right represent examples of partial digests of the polyoma genome with HpaII. The middle gel shows the fragments generated by partial digestion of the superhelical circular DNA, and the gel on the right shows the HpaII fragments formed after the circular genome is converted into a linear molecule by EcoR1 (an enzyme that makes only one cut in the circle). (B) The restriction map of the linearized polyoma genome based on cleavage by HpaII. The eight fragments from the complete digest are shown along the DNA at the top. The overlapping fragments from the partial digest are shown in their ordered arrangement below the map. (Fragments L and M migrate to the bottom of the gel in part A, right side.)

Figure 18.38Formation of a recombinant DNA molecule. In this example, a preparation of bacterial plasmids is treated with a restriction enzyme that makes a single cut within each bacterial plasmid. This same restriction enzyme is used to fragment a preparation of human genomic DNA into small fragments. Because they have been treated with the same restriction enzyme, the cleaved plasmid DNA and the human DNA fragments will have sticky ends. When these two populations are incubated together, the two DNA molecules become noncovalently joined to each other and are then covalently sealed by DNA ligase, forming a recombinant DNA molecule.

Figure 18.39An example of DNA cloning using bacterial plasmids. DNA is extracted from human cells, fragmented with EcoR1, and the fragments are inserted into a population of bacterial plasmids. Techniques are available to prevent the formation of plasmids that lack a foreign DNA insert. Once a bacterial cell has picked up a recombinant plasmid from the medium, the cell gives rise to a colony of cells containing the recombinant DNA molecule. In this example, most of the bacteria contain eukaryotic DNAs of unknown function (labeled as ?), while one contains a portion of the DNA encoding ribosomal RNA, and another contains DNA encoding insulin.

Figure 18.40a, bLocating a bacterial colony containing a desired DNA sequence by replica plating and in situ hybridization. (A) Once the bacterial cells that were plated on the culture dish have grown into colonies, the dish is inverted over a piece of filter paper, allowing some of the cells from each colony to become adsorbed to the paper. Empty culture dishes are then inoculated by pressing them against the filter paper to produce replica plates. (B) Procedure for screening the cells of a culture dish for those colonies that contain the recombinant DNA of interest. Once the relevant colonies are identified, cells can be removed from the original dish and grown separately to yield large quantities of the DNA fragments being sought.

Figure 18.40a, bLocating a bacterial colony containing a desired DNA sequence by replica plating and in situ hybridization. (A) Once the bacterial cells that were plated on the culture dish have grown into colonies, the dish is inverted over a piece of filter paper, allowing some of the cells from each colony to become adsorbed to the paper. Empty culture dishes are then inoculated by pressing them against the filter paper to produce replica plates. (B) Procedure for screening the cells of a culture dish for those colonies that contain the recombinant DNA of interest. Once the relevant colonies are identified, cells can be removed from the original dish and grown separately to yield large quantities of the DNA fragments being sought.

Figure 18.41Separation of plasmid DNA from that of the main bacterial chromosome by CsCl equilibrium centrifugation. This centrifugation tube can be seen to contain two bands, one of plasmid DNA carrying a foreign DNA segment that has been cloned within the bacteria, and the other containing chromosomal DNA from these same bacteria. The two types of DNA have been separated during centrifugation. The researcher is removing the DNA from the tube with a needle and syringe. The DNA in the tube is made visible using the DNA-binding compound ethidium bromide, which fluoresces under ultraviolet light.

Figure 18.42Protocol for cloning eukaryotic DNA fragments in lambda phage. The steps are described in the text.

Figure 18.43Polymerase chain reaction (PCR). As discussed in the text, the procedure takes advantage of a heat-resistant DNA polymerase whose activity is not destroyed when the temperature is raised to separate the two strands of the double helix. With each cycle of duplication, the strands are separated, flanking segments (primers) bind to the ends of the selected region, and the polymerase copies the intervening segment.

Figure 18.44a-cDNA sequencing. (A) The basic steps in sequencing a small hypothetical fragment by the Sanger-Coulson (dideoxy) technique, as described in thetext. (B) Gel lanes in which fluorescently labeled daughter molecules have been separated. The color of the band is determined by the identity of the dideoxynucleotide at the 3 end of the DNA strand. (C) The sequence of nucleotides in the template strand is interpreted by a computer that reads from the bottom to the top of the gel, using the intensity and wavelength of the fluorescent light as input. The computer generates an electropherogram showing the intensity and color of the detected fluorescence, along with the DNA sequence interpretation.

Figure 18.44a-cDNA sequencing. (A) The basic steps in sequencing a small hypothetical fragment by the Sanger-Coulson (dideoxy) technique, as described in thetext. (B) Gel lanes in which fluorescently labeled daughter molecules have been separated. The color of the band is determined by the identity of the dideoxynucleotide at the 3 end of the DNA strand. (C) The sequence of nucleotides in the template strand is interpreted by a computer that reads from the bottom to the top of the gel, using the intensity and wavelength of the fluorescent light as input. The computer generates an electropherogram showing the intensity and color of the detected fluorescence, along with the DNA sequence interpretation.

Figure 18.45a, bSynthesizing cDNAs for cloning in a plasmid. (A) In this method of cDNA formation, a short poly(dT) primer is bound to the poly(A) of each mRNA, and the mRNA is transcribed by reverse transcriptase (which requires the primer to initiate DNA synthesis). Once the DNARNA hybrid is formed, the RNA is nicked by treatment with RNase H, and DNA polymerase is added to digest the RNA and replace it with DNA, just as it does during DNA replication in a bacterial cell. (B) To prepare the blunt-ended cDNA for cloning, a short stretch of poly(G) is added to the 3 ends of the cDNA and a complementary stretch of poly(C) is added to the 3 ends of the plasmid DNA. The two DNAs are mixed and allowed to form recombinants, which are sealed and used to transform bacterial cells in which they are cloned.

Figure 18.46Microinjection of DNA into the nucleus of a recently fertilized mouse egg. The egg is held in place by a suction pipette shown at the right, while the injection pipette is shown penetrating the egg at the left.

Figure 18.47Transgenic mice. This photograph shows a pair of littermates at age 10 weeks. The larger mouse developed from an egg that had been injected with DNA containing the rat growth hormone gene placed downstream from a metallothionein promoter. The larger mouse weighs 44 g; the smaller, uninjected control weighs 29 g. The rat GH gene was transmitted to offspring that also grew larger than the controls.

Figure 18.48Formation of transgenic plants using the Ti plasmid. The transgene is spliced into the DNA of the Ti plasmid, which is reintroduced into host bacteria. Bacteria containing the recombinant plasmid are then used to transform plant cells, in this case cells of the meristem at the tip of a dissected shoot apex. The transformed shoots are transferred to a selection medium where they develop roots. The rooted plants can then be transferred to potting soil.

Figure 18.49Formation of knockout mice. The steps are described in the text.




Figure 18.50Determining gene function by RNA interference. The figure shows a gallery of immunofluorescence images of cultured Drosophila cells that had been incubated with various double-stranded siRNAs. The cells shown here had accumulated in metaphase of mitosis as the result of a separate treatment that blocked progression into anaphase. During the course of this study, over 4 million cells were examined. It was possible to screen such a large number of cells by having a computer select the images of cells that were in metaphase and then crop and assemble the images into panels as shown in this photograph. The images could then be screened for the presence of abnormal mitotic spindles either by human observers or by computer analysis using programs designed to detect specific features of an abnormal spindle.

Figure 18.51Determining gene function by RNA interference. (Left) An untreated, cultured mammalian cell at metaphase of mitosis. (Right) The same type of cell that has been treated with a 21-nucleotide dsRNA designed to induce the destruction of mRNAs encoding Aurora B kinase, a protein involved in the metaphase spindle checkpoint. The chromosomes in this cell lie adjacent to the mitotic spindle, suggesting the absence of kinetochoremicrotubule interactions.

Figure 18.52Formation of monoclonal antibodies. The steps are described in the text. The HAT medium is so named because it contains hypoxanthine, aminopterin, and thymidine. This medium allows cells with a functional hypoxanthineguanine phosphoribosyl transferase (HGPRT) to grow, but it does not support the growth of cells lacking this enzyme, such as the unfused myeloma cells used in this procedure.

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