Chapter 18: Microscopes, Cell Culture, Proteins, Nucleic Acid, DNA, Antibodies

Ion exchange, gel-filtration(size-exclusion) and affinity chromatography

-based on surface density, may be diluted differently -the larger the molecules the less time it spends in the beats, the big ones will come out early (size exclusion) -specific ligand for affinity chromatography

Maximum magnification using a scanning electron microscope is about:

150,000x

18.15 Proteins and peptides by Mass Spectrometry

Principles of operation of a mass spectrometer: -Protein Measurement and Analysis -The 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 -allows you to read out the mass charge on the protein

18.10 Physical Fractionation of Cell's Contents by Differential Centrifugation

Purification 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. Differential 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 activities -form gradient in a special form such that individual components in the cell are separated In a density gradient equilibrium centrifugation a continuous sucrose-density gradient different organelles sediment according to density, where they form bands.

DNA fragments and DNA probes are to Southern blotting as ________ are to Northern blotting.

RNA fragments and DNA probes

Which of the following statements is NOT true regarding green fluorescent protein (GFP)? A: It occurs naturally in jellyfish and other organisms that flow in the dark. B: It can be used to follow the activities of tagged molecules in living cells. C: The gene that encodes GFP has been incorporated into the DNA of other organisms and expressed as fluorescence. D: GFP does not require an additional cofactor to absorb and emit light. E: Research into cloning this gene for molecular biological studies is not yet complete.

Research into cloning this gene for molecular biological studies is not yet complete

18.5 Specimen Preparation for Electron Microscopy **image ppt 3 slide 14**

Specimen Preparation for Electron Microscopy: -Specimens 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.

18.4 LM and TEM use similar optical principles

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.

Which of the following statements is TRUE regarding 2d generation sequencing? A: They directly identify individual nucleotides as they are being incorporated into DNA in real time. B: Fragments that differ by only one nucleotide are separated by high-resolution gel electrophoresis. C: DNA fragments are pulled through a nanopore and identified one nucleotide at a time. You got it wrong' D: They are the best way to determine the DNA sequence of a new species. E: None of these statements are true regarding 2d generation sequencing.

They directly identify individual nucleotides as they are being incorporated into DNA in real time.

18.24 Transgenic Eukaryotic Cells and Mammalian Embryos

Transduction: DNA into a virus Transformation:DNA into bacteria Transfection : DNA into a culture cell Transgenic animals (or plants) Microinjection 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. Transgenic 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. -DNA 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. -insert dna into host genome, select cells, and move them to animal to reproduce whole organisms

A genomic library is

a collection of DNA fragments that make up the entire genome of a particular organism

A "gene gun" is

a gun that fires DNA-coated pellets into plant cells

Which technique would be best to study integral proteins in the cell membrane?

freeze-fracture replication

Which type of column chromatography separates proteins on the basis of molecular weight?

gel filtration chromatography

Ultracentrifuges can generate centrifugal forces ________ the force of gravity.

greater than 500,000 times

The main benefit of a three-dimensional culture system is that:

the morphology and behavior of cells is closer to what they would have within the body.

You have homogenated and fractionated rat liver cells using differential centrifugation. Which fraction would have the fewest mitochondria?

the supernatant that results after a high-speed ultracentrifugation step

A scanning electron microscope is the instrument of choice when you want to study:

the surface of small structures

Scanning electron microscopy is different from transmission electron microscopy in that:

you can view larger specimens and electrons do not penetrate the specimen

18.9 Cell Culture

-A primary culture is when cells are obtained directly from an organism. -A secondary culture is derived from a primary culture into long-term growing cells. -A cell line refers to cells with genetic modifications that allow them to grow indefinitely. Most 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. -Many types of plant and animal cells can be grown in culture. -cell line is immortalized, can live forever

light microscope has an objective lens with a magnification of 40x and an ocular lens with a magnification of 10x. What is the total magnification of the image?

400x

18.2 Bright-Field , Phase-Contrast, and Interference Contrast **pics of diffs ppt 3 slide 6**

A 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. Phase-Contrast Microscopy: The 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.

18.1 The Light Microscope **images ppt 3 slide 2**

A microscope that has both an objective and an ocular lens. The 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. The 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. -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. -light source and condenser that makes it relatively even and then a specimen is shined and objective lens forms the image (source, object, imaging)

18.26 Antibodies against specific antigens

Antibodies 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. -antibody can be labeled and give you the signal for detection -get human insulin and inject into mice, mice will produce antibodies, induce cell fusion, create hybridomia, can continue to produce antibody, if you get rid of medium you will kill parent cells but hybrid will move on

18.19 Chemical Synthesis of DNA or RNA

Automated chemical reactions to link nucleotides in a linear fashion Oligos up to a total of 100 nucleotides can be produced in each processive run Chemical modifications can be introduced to specific positions. Longer sequences can be produced from synthetic segments.

18.11 Biochemical separations by Liquid Column Chromatography

Biochemical Separation is usually achieved based on differential interactions between the target molecules and the contaminants with the matrix materials. such as: -ion-ion interactions -motion of molecules within a porous matrix -ligand-receptor recognition -pH-dependent changes in interactions

Protein Cas9, the RNA‐guided DNA endonuclease that is used in CRISPR‐based genome editing

Cas9 protein (purple) associates with a guide RNA (purple backbone with white bases) and scans along genomic DNA looking for a segment of DNA (blue backbone with white bases) that matches with the guide RNA. When it finds this match, it cleaves the DNA at that point.

Nucleic acid hybridization can be used as a measure of evolutionary relationships between species. Of the following statements about the DNA of related species, which is true?

Closely related species form hybrid DNAs with relatively high melting temperatures.

18.16 Structure determination by nuclear magnetic resonance (NMR) spectrometry

Combining data from electron microscopy and X-ray crystallography: provides information on protein-protein 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. 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. -every molecule has a signature map

The heat-stable enzyme used in the polymerase chain reaction is a type of

DNA polymerase

Visibility-contrast in images **image ppt 3 slide 5**

Feulgen stain: specific for DNA as indicated by the localization of the dye, showing the chromosomes of an onion root tip cell in metaphase of mitosis at the time it was fixed Visibility: Light passing, Contrast due to light scattering or light absorption, Preparation of Specimens for Bright-Field Light Microscopy: -A whole mount is an intact object, either living of dead. -A section is a very thin slice of an object: 1. To prepare a section, cells are immersed in a chemical called a fixative. 2. The rest of the procedures minimize alteration from the living state. -chromosome in prophase of cell division

18.7 AFM: Atomic Force Microscopy

High-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 sample's 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. Atomic 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. -sensitive to small changes at the surface of the sample -allows to obtain high resolution -radio isoptopes for labeling High-speed AFM: Forces between the sample and the AFM tip cause deflections of the tip, which are detected by a laser beam. AFM images of a living endothelial cell in plastic petri dish using the JPK NanoWizard

Which of the following statements is NOT true regarding cryofixation? It minimizes the formation of artifacts. B: It is used by medical personnel to examine tissues during surgery. C: If performed too quickly, it can result in the formation of ice crystals that destroy the appearance of the cell. D: It may employ high pressure and liquid nitrogen. E: Temperatures below the freezing point of water need to be employed.

If performed too quickly, it can result in the formation of ice crystals that destroy the appearance of the cell.

18.26 The Use of Antibodies

Immunoprecipitation Western blotting in protein gels (specific epitopes in proteins) Immunohistological location of antigens Fluorescent antibodies for FM examination Indirect labeling with secondary antibodies for visualization 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.

You are interested in reconstructing the three-dimensional shape of chromosomes in living cells that are in the process of mitosis. Which microscopic technique will you choose?

Laser scanning confocal microscopy

Which of the following does NOT describe the relationship between light microscopy and electron microscopy? A: Electron microscopes are capable of higher magnification than light microscopes. B: Light microscopes can be used to view living cells, but electron microscopes cannot. C: Electron microscopes have greater resolution than light microscopes. D: Light microscopes use shorter wavelengths than electron microscopes. This is correct answer E: Electron microscopes do not detect color, but light microscopes can

Light microscopes use shorter wavelengths than electron microscopes.

You think you might have discovered a new organelle using electron microscopy, but it might be an artifact of your technique. What might you do to prove it is real?

Look for the same structure in specimens prepared using different (or no) fixatives.

Resolution **images ppt 3 slide 3**

Magnification 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. The 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. -human eye sensitive to color and intensity changes

18.14 Spectrometric property of proteins and nucleic acids

Optical spectrometer : light absorption by materials (Abs spectra) de Beer's law Mass spectrometer --- mass-to-charge ratio and abundance of gas-phase ions. Time-of-flight spectrometer --- time of flight between two detectors; establishing velocity and mass/charge relation. Magnetic spectrometer --- force of a charged particle in a magnetic field

18.16 Structure Determination by cryoEM imaging of single macromolecular complexes

Overview of single particle cryo‐EM reconstruction: After a sample is prepared, a small volume of the sample is placed on a carbon grid, which is gently blotted to remove excess liquid and rapidly plunged into liquid ethane. The grids are then viewed through a transmission electron microscope, preferably fitted with a direct electron detector. From the images obtained, many thousands of particles are selected and placed into classes that can represent different sample orientations or states. The data is processed computationally, and a 3D density map is constructed, which reveals the structure of the protein. With recent advancements in technology and software, atomic‐level structures have been solved using cryo‐EM. -sample is prepared and imaged in freezing conditions to obtain structure 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.

18.22 DNA amplification by Polymerase Chain Reaction (PCR)

Polymerase 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. Polymerase 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. -can quickly amplify individual molecules; PCR is used to characterize DNA signatures of human beings; majority have different ones bc of mutations PCR can be used for amplification, verification, and quantification of a target DNA fragment.

18.25 RNA Interference (RNAi) for gene silencing

RNAi for genome-wide silencing of genes The cells are accumulated in metaphase of mitosis. -Determining 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. RNA Interference -Specific 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. -diff rna for diff genotypes

18.3 Super-Resolution FM beyond diffraction limits

STORM as an example. Other modes include PALM, STED, etc Breaking the light microscope's 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. -using comp to centralize individual molecules and then put them together to achieve high resolution -molecular details much better resolved using this microscope Super-Resolution Fluorescence Microscopy: -STORM (stochastic optical reconstruction microscopy) allows the localization of a single fluorescent molecule within a resolution of <20 nm. -Fluorescent images can be positioned with greater accuracy. -Several distinct conceptual strategies have been introduced to overcome the diffraction barrier and enable the analysis of biological structures at the superresolution level. One highly developed strategy, often referred to as point-spread function engineering or illumination-based superresolution, utilizes non-linear optical approaches to reduce the focal spot size. Examples of this type of superresolution imaging are stimulated emission depletion (STED), ground state depletion (GSD), and saturated structured illumination (SSIM) microscopy. -A second (and increasingly popular) strategy for overcoming the diffraction barrier employs photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution. This approach relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. The imaging methods using probe-based superresolution include photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM).

18.6 SEM: Scanning Electron Microscopy

Scanning electron micrographs of (A) a T4 bacteriophage (X 275,000) and (B) the head of an insect (X 40). Scanning 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 -this mode allows you to see the surface of the sample

18.18 Nucleic Acid Hybridization

Southern blot technique --> DNA-DNA hybridization. Northern blot technique --> RNA-DNA hybridization. Determining 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. 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. -can be transferred from gel to membrane and then can be stained by specific labeled probes

18.21 Recombinant DNA Technology

The 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.) -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. -can cut out a piece of DNA from one source and insert into a plasmid that allows you to propogate these fragments -genome dna library to preserve fragments

Under which of the following conditions would the visibility of an object under a microscope be lowest

The object has the same refractive index as does the medium.

Why are heat-stable DNA polymerases from thermophilic bacteria required for the polymerase chain reaction?

These enzymes are stable enough to withstand the temperatures required to melt DNA.

18.13 Thousands of Proteins separated by 2D Polyacrylamide Gel Electrophoresis

Two-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. Two-Dimensional Gel Electrophoresis: -It 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. -example of autoradiography -2 dimensionally separated based on 2D weight

18.3 Fluorescence Microscopy (FM)

Using fluorophores, such as chemicals or proteins, to mark specific molecules. 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.

Which of the following statements is NOT true regarding cell-free systems? A: They contain organelles isolated by differential centrifugation. B: They contain organelles that no longer function. C: They can be used to study DNA synthesis. D: They can be used to study membrane transport. E: Whole cells may be reconstituted from them.

Whole cells may be reconstituted from them.

18.16 Structure determination by X-ray crystallography

X-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. 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. -grow a crystal and obtain diffraction pattern through xray beam

The key difference between a primary cell culture and a secondary cell culture is:

a primary cell culture is obtained directly from the cells of an organism; a secondary cell culture is derived from a previous culture.

A fluorophore

absorbs light at one wavelength and emits a portion of the light at a different wavelength

Which of the following would be the LEAST promising source of cells for a primary cell culture?

adult rat brain tissue

Which type of chromatography can use the ligand-binding properties of a protein to produce a nearly pure product in a single step?

affinity chromatography

Once the nucleotide sequence of a DNA segment has been determined: A: it can be compared to the DNA sequence of other organisms to assess their genetic relatedness. B: it can be compared to the sequence of other organisms of the same species to determine individual genetic differences. C: it can be used to determine the amino acid sequences of proteins that it encodes. D: it can be used to predict the structure, location and possible function of proteins within a cell. E: All of the choices are correct

all of the above

YACs differ from BACs in that they

are inserted into yeast cells and BACs are inserted into E. coli

You want to know if a culture of cells is in the process of DNA synthesis. You incubate your cells in the presence of radioactive thymidine to see if it is being incorporated into the DNA. What is the best technique to detect the labeled deoxynucleotide in nuclear DNA?

autoradiography

The role of the vector DNA in DNA cloning is to:

both identify the host cell that has taken up the one specific gene of interest and carry the foreign DNA into the host cell.

cDNA lacks

both promoter sequences and introns

Phase-contrast microscopes are very useful for viewing:

both transparent objects and living cells

Atomic force microscopy is LEAST similar to

cryo-EM because the specimen must be frozen

Restriction endonucleases function to:

cut stretches of DNA at specific sequences.

Diffraction limit in resolution

d= (0.61 lambda)/(n sin(alpha) ) -if center of a diffraction spot is located at first diffraction minimum of another spot Limit of resolution by visible light d= (0.61 lambda)/(n sin(alpha) ) = (0.61 lambda)/NA = (0.61 * 400 nm)/1.2 = 200 nm d = minimum distance that 2 points in specimen must be separated by in order to be resolved l = the wavelength of light (527 nm used for white light) n = refractive index (RI) of medium present between the specimen & the objective lens 4. a = half the angle of cone of light entering objective lens (angle between normal to base of light cone & its side); a is a measure of light-gathering ability of lens; directly related to its aperture -the shorter the wavelength, the better the resolution

Once an electrophoretic gel has been removed from its container, the proteins may be analyzed by all techniques EXCEPT: A: their interaction with specific antibodies in a blot. B: staining them. C: attaching radioactive labels that can indicate positions on the gel by producing an autoradiograph. D: determining the net charge on each protein. This is correct answer E: slicing the gel into fractions and isolating the proteins.

determining the net charge on each protein

Which of the following statements is TRUE regarding the synthesis of polynucleotides? A: It was begun by Khorana in 1981. B: The gene for human interferon was the first polynucleotide to be synthesized. C: A computer-controlled machine assembles a polynucleotide one nucleotide at a time from the 5′ end to the 3′ end. D: Flurophores can be incorporated into the synthesized polynucleotide. E: Double-stranded polynucleotides can be produced by the computer-controlled machine.

flurophores can be incorporated into the synthesized polynucleotide

The yeast two-hybrid system takes advantage of the activation of __________________ to reveal protein-protein interactions.

gene expression

Max Perutz solved the structure of ___________ using x-ray diffraction

hemoglobin

Which of the following is NOT a step in preparation of specimens for bright-field light microscopy? A: dehydration in a series of alcohols B: application of a fixative that penetrates the cell membrane and maintains cell structure C: embedding in paraffin D: immersion in toluene E: initial rinse in water of the fresh specimen under study

initial rinse in water of the fresh specimen under study

In electrophoresis, the tracking dye:

moves faster than the proteins being analyzed.

The first protein to be analyzed by x-ray diffraction was:

myoglobin

Which of the following is NOT a feature of analysis with mass spectrometry? A: magnets B: positively charged gaseous ions C: a positively charged plate You got it right D: a curved tube E: a negatively charged plate

positively charged plate

The function of DNA cloning is to:

produce large quantities of a specific DNA segment

18.12 Protein interactome: Protein-Protein Interactions

-Co-precipitation -yeast two-hybrid system -Membrane-based interaction detection -GFP-based interaction detection Use 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 domain—are seen here as the transcription factor binds to the promoter of a gene ( lacZ) encoding β-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. -A DNA binding domain is linked to the gene for one protein—the "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. -when bait and fish are together you can detect a signal -proteins specific for your bait

18.25 Gene Editing and Silencing Mutagenesis for generating knockout mice

-Forward genetics: from phenotype to genotype -Reverse genetics: from genotype to phenotype -This 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 genotype In Vitro Mutagenesis: -Site-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.

18.3 Laser Scanning Confocal Microscopy

-Pinholes used to select a specific point in a section Laser 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 μm 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. The light paths in a confocal fluorescence microscope and the focal plan: -A 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 -sample needs to be transparent -manipulate system to scan across x y direction, can manipulate the focus level

18.13 Protein run in polyacrylamide Gel Electrophoresis

-SDS-binding keeps proteins denatured, evenly negatively charged and linear. -Electrophoresis: separation in electrical field based on charge density and MW. Detection by . Staining by stains . Antibody-based for western blot (chemiluminescence or fluorescence) . Fluorescence . Radioactivity Polyacrylamide 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). -sds binding denatures protein and makes it evenly charged to separate in the electric field, protein goes from negative side -can be stained by specific antibody and labeled by fluroescents Electrophoresis 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.

Oligonucleotides can be synthesized by a computer-controlled machine up to lengths of about how many nucleotides?

200

The half-life of tritium is 12 years. What percentage of a radioactive sample of tritium would remain after 24 years?

25%

18.23 DNA Libraries

DNA 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 Vectors -A 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 libraries cDNA libraries gDNA libraries YAC BAC

18.20 DNA Sequencing

DNA sequencing: (A) The basic steps in sequencing a small hypothetical fragment by the Sanger-Coulson (dideoxy) technique, as described in the text. (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. Techniques 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. Next generation-can read out thousands of short segments in a single round

Which of the following statements is NOT true regarding a yeast two-hybrid system? A: This technique can investigate whether proteins will interact with one another. B: A functional transcription factor is created when two different proteins interact with one another and activate a reporter gene. C: The activity of a reporter gene can be used to monitor the presence of an active transcription factor. D: Either hybrid protein produced in this system can activate the reporter gene, but if the proteins interact, the reporter gene is turned off. E: Large numbers of proteins can be screened using this system.

Either hybrid protein produced in this system can activate the reporter gene, but if the proteins interact, the reporter gene is turned off.

18.8 Radioisotopes in Cell Biology

Energy from radioactive particles are converted into light (photons) for detection: Liquid scintillation to count the isotope elements Autoradiography to localize the radio-labeled molecules Radioisotopes 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. -Liquid scintillation spectrometry -Scintillants 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. 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. (Kv4.x data showing the northern blot results for total mRNA prep from adult rat brain.) SILAC: stable isotope labeling by amino acids in Cell Culture.

18.5 Enhance contrast for TEM specimens

Examples 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). -The 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. -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. -meta shadowing: takes advantage of 3D shape -shattering does not allow you to see interior of sample

18.3 Light Sheet FM (4D imaging)

Examples of samples visualized using lattice light sheet microscopy.: -Lattice light sheet microscopy utilizes several parallel thin 2D light sheets (blue/green lines in center image) that sweep across a sample, resulting in 3D images of fluorophore emission. This method allows for rapid imaging in 3D over long periods of time and has been used to investigate a variety of systems, including the dynamics of cell division (upper left, bottom center) and cellular motility (center, lower right). Light-Sheet Fluorescence Microscopy: -Visualize the subcellular localization of a protein of interest in a fly or worm embryo at subsecond intervals. -The ability to collect high-resolution images in 4D (three dimensions and time) -It is anticipated that lattice light sheet microscopy will provide numerous insights into how dynamic molecular processes occur in living cells. Ultrathin Light-sheet FM: http://science.sciencemag.org/content/346/6208/1257998

Which of the following statements is NOT true regarding a cDNA library? A: It is derived from messenger RNAs that are present in a cell. B: It contains all of the DNA sequences of a species. C: It is a collection of cloned DNA fragments. D: It contains DNA for the genes that are active within a cell. E: It cannot be directly compared to the sequences found in genomic libraries.

It contains all of the DNA sequences of a species.

18.5 TEM: Freeze-Fracture Replica and Freeze-Etching

Procedure 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. -In 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.

18.25 Genome Editing Using Engineered Nucleases

Overview of the CRISPR technique of genome editing. Cas9 (blue) utilizes a guide RNA (purple) that can recognize and basepair with a target genomic sequence (thick red strands), Cas9 also requires a short sequence, known as a PAM sequence (orange), downstream for the target sequence. Upon finding a matching sequence, Cas9 cuts both strands of target DNA, resulting in a double‐stranded break which is rapidly repaired by DNA repair enzymes. CRISPR can be used to edit, delete, or add new DNA to a specific location in the genome. CRISPR was discovered for its role in bacterial innate immunity; some species take up segments of the phage DNA and incorporate them into their own genome: -The DNA can be transcribed into RNA and incorporated into Cas9 protein, a nuclease that uses the short phage sequences in its RNA as a template to identify and destroy phage DNA that it encounters in the cell through DSBs. -By introducing lab designed RNAs to Cas9, the nuclease could identify and cut DNA of arbitrary sequence with high specificity and efficiency. -select for mutations and produce an organism with them

18.17 Fractionation of Nucleic Acids by Gel Electrophoresis

Separation 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. Separation 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. -analyze by electrophoresis and separate them by size

18.4 Transmission Electron Microscopy (TEM)

TEM has much better resolution than LM -wavelength of elctrons at > 100 kV: ~<0.005nm d= (0.61 lambda)/(n sin(alpha)) -A 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 μm, 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 μm 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. Transmission electron microscopes (TEMs) use electrons instead of light to form images: -The limit of resolution is about 10-15 Å. -The resulting images display a 100- to 200-fold increase in resolution over traditional light microscopy. -Increased amount of information at the subcellular level. -means electron goes through the sample -super small wavelength -not good resolution from light microscopy but here you do and can see individual ribosomes with TEM

18.17 Fractionation of Nucleic Acids by Ultracentrifugation

Techniques 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 tube's contents. The absorbance of the solution in each fraction is measured and plotted as shown. Ultracentrifugation: -Velocity 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.

The beam of electrons in a TEM can be focused by

electromagnets

Which method begins with production of a continuous density gradient using a salt of cesium?

equilibrium centrifugation

Which of the following does NOT describe a potential use of atomic force microscopy? A: measurement of affinity of a molecule for a ligand B: determination of molecular structure C: study of movement of molecules D: analysis of the mechanical properties of cell components E: examination of the profiles of organelles within a fractured cell

examination of the profiles of organelles within a fractured cell

The polymerase chain reaction (PCR) can be used for all of the following EXCEPT: A: determining whether a tissue sample contains a particular virus. B: amplifying the amount of DNA obtained from bloody clothing. C: analyzing the similarity of two DNA molecules. D: measuring how much of a specific nucleotide sequence is in a sample. E: extending the length of a piece of amplified DNA.

extending the length of a piece of amplified DNA.

Which of the following does NOT contribute to the resolution attainable by a microscope? A: the wavelength of the light B: the magnification of the objective lens C: the refractive index of the material between the specimen and the lens D: the numerical aperture of the lens E: all of these contribute to resolution

the magnification of the objective lens

In liquid chromatography, the more that a protein molecule interacts with the materials of the matrix:

the more slowly it passes through the column

Radioactive molecules can be used as tracers because of all of the following EXCEPT: A: their exceptionally long half-lives contribute to stability. This is correct answer B: they participate in the same chemical reactions as their nonradioactive counterparts. C: they are available as isotopes of common atoms found in living systems. D: they can be administered in low doses that cause minimal damage to living tissue. E: they emit energy that can be detected by an instrument.

their exceptionally long half-lives contribute to stability