Biology Module 2
How does differential interference contrast microscopy (Nomarski) work? (LM)
As in phase-contact microscopy, optical modifications are used to exaggerate differences in density; the image appears almost 3-D.
In plant cells, what is the plasmodesmata, what is it made up of, and what does it do?
Cytoplasmic channels thru cell walls that connect the cytoplasms of adjacent cells
In plant cells, what is the central vacuole, what is it made up of, and what does it do?
Prominent organelle in older plant cells; functions include storage, breakdown of waste products, and hydrolysis of macromolecules; enlargement of the vacuole is a major mechanism of plant growth
What happens to lysosomal enzymes if the lysosome breaks open?
Some leakage: the released enzymes are ineffective cuz the cytosol has a more neutral pH Excessive leakage: destroy a cell by self-digestion
Fluorescence microscopy using fluorescent molecules attached to antibodies and GFP fluorescence allows the visualization of cell structures composed of distinct proteins. Which of the following experiments can be conducted on living cells? a. GFP visualization of mitochondrial movement in living cells. b. Fluorescent antibody detection of chromosome segregation in living cells. c. Scanning electron microscope imaging of fluorescent red cells moving through capillaries. d. GFP visualization of insulin secretion in thin section electron micrographs.
a. GFP visualization of mitochondrial movement in living cells.
What are microvilli?
fingerlike extensions of the plasma membrane that increase tissues surface area.
What is the GFP gene?
green fluorescent protein If you have the gene for this and you could stitch that gene on to a gene that encodes a protein of interest, you would have a built-in tag that would allow your protein of interest to be tracked in a living cell. These cells didn't have to be killed because the fluorescence was put in place by genetic engineering. The structure, the three-dimensional atomic resolution structure of the green fluorescent protein was shown to reveal these beta strands in a kind of a barrel aligned around an interior where tryptophan residues are aligned so that this molecule has intrinsic fluorescence. It absorbs ultraviolet light of a particular wavelength and emits visible light.
What does cell fractionation enable?
scientists to determine the functions of organelles. One test showed presence of enzymes in cellular respiration
What is a lysosome?
membranous sac of hydrolytic enzymes that can digest (hydrolyze) macromolecules Lysosomal enzymes work best in acidic environment found in lysosomes.
What is brightfield microscopy (light microscopy, LM)?
when dark objects are visible against a bright background. In brightfield, light passes directly through the specimen. When unstained (left image), the image has little contrast, but staining (right image) the dyes enhances the contrast. Most strains require cells to be preserved, which kills them.
How many chromosomes does a human cell have?
46. Sex cells only have 23
What is the direct relationship among the organelles of the endomembrane system?
1) The nuclear envelope is connected to the rough ER which is also cont. w/ the smooth ER 2) Membranes and proteins produced by the ER move via transport vesicles to the Golgi 3) The golgi pinches off transport vesicles and other vesicles that give rise to lysosomes; other types of specialized vesicles, and vacuoles 4) The lysosome is available for fusion w/ another vesicle for digestion. 5) A transport vesicle carries proteins to the plasma membrane for secretion. 6) The plasma membrane expands by fusion of vesicles; proteins are secreted from the cell by exocytosis.
What is the vesicle's path through the Golgi apparatus?
1) Vesicles move from ER to golgi 2) vesicles coalesce to form a new cis Golgi cisternae 3) Cisternal maturation: golgi cisternae move in a cis to trans direction 4) Vesicles form and leave Golgi, carrying specific products to other locations or to the plasma membrane for secretion 5)Some vesicles transport some proteins backward to less mature golgi cisternae, where they function 6) some vesicles also transport certain proteins back to ER
What four things do all types of cells have?
1) plasma membrane 2) cytosol 3) Chromosomes (carry genes in DNA form) 4) ribosomes (complexes that make proteins based on gene instruction)
What does the plasma membrane and organelle membranes consist of?
A double layer (bilayer) of phospholipids w/ various proteins attached to or embedded in it. Hydrophobic parts are in the interior of the membrane, and vice versa. Carbohydrate side chains may be attached to proteins or lipids on the plasma membrane's outer surface.
How does chromatin appear when a cell is not dividing and dividing?
A mass that cannot be distinguished from one another, even though discrete chromosomes are present. When preparing to divide, the chromosomes form loops and coil, condensing and becoming thick enough to become visibly distinguishable
What are fimbriae, glycocalyx, and flagella?
All are only found in prokaryotes. Fimbriae: attachment structures on the surface of some prokaryotes (not visible on TEM) Glycocalyx: outer coating of many prokaryotes, consisting of a capsule or a slime layer Flagella: locomotion organelles of some prokaryotes
What are vacuoles?
Large vesicles derived from the ER and Golgi apparatus. Diverse maintenance compartments
What is phagocytosis?
Amoebas and unicellular protists eat by engulfing smaller organisms or food particles. A food vacuole forms and then fuses w/ a lysosome, whose enzymes digest the food.
Why is yeast a particularly favorable organism to use for exploring gene function?
Because it can grow in a haploid phase, meaning that it grows as a cell with a single copy of each gene. So when you expose a yeast cell to a chemical that introduces mutations in the genes randomly, that mutation may be expressed very early after the mutation has been introduced. On the other hand, if you have a cell that has two copies of every gene (humans), it becomes more difficult to do traditional genetics because when you expose a cultured human cell to a chemical that causes mutations, the mutation that affects a gene will not become apparent unless both copies of that gene had been hit by the same mutation.
How are hydrolytic enzymes and lysosomal membranes made?
By the rough ER and then transferred to the Golgi apparatus for further processing.
What are the faces of the golgi apparatus and what do they do?
Cis: (near ER) receives vesicles containing ER products when vesicle from ER fuses w/ a golgi membrane trans: dispatches vesicles Cisternae move from cis to trans while carrying some protein along (some move backward)
In lecture we discussed the creation of temperature sensitive lethal yeast mutants. What is the benefit of using such mutants, and which types of genes would be best probed using them?
Conditionally lethal mutants allow for the interrogation of essential genes. In the case of the temperature sensitive yeast mutants, they allowed researchers to identify genes involved in essential cellular processes such as budding and cell division -- simple knockouts of these genes would result in non-viable cells.
What is the experimental goal of X-ray crystallography?
Determining the atomic structure of a macromolecule
In animal cells, what is the lysosome, what is it made up of, and what does it do?
Digestive organelle where macromolecules are hydrolyzed
What are chromosomes?
Discrete units of DNA in the nucleus that carry the genetic info. Each chromosome has one long DNA molecule associated w/ many proteins, including small basic proteins called histones.
Where is DNA located in pro and euk cells?
E: most DNA is in nucleus (double membrane) P: concentrated in a non membrane enclose region called a nucleoid, not as much space cuz pro are much smaller than euk
What is the experimental goal of Immunofluorescence?
Examining the localization of insulin within a cell
What is the experimental goal of Cell fractionation?
Examining the localization of insulin within a cell Separating a protein of a known size
What is the experimental goal of electron microscopy?
Examining the localization of insulin within a cell Examining the topography of a cell
What is the experimental goal of Super resolution light microscopy?
Examining the localization of insulin within a cell Examining membrane fusion at the edge of a cell in real-time Examining the topography of a cell
What is the experimental goal of GFP fusion protein?
Examining the localization of insulin within a cell and examining membrane fusion at the edge of a cell in real-time
What is the nuclear matrix?
Framework of protein fibers extending throughout the nuclear interior. May help organize genetic material
Green Fluorescent Protein (GFP) and immunofluorescence both take advantage of fluorescence to study cells and cell function, however these techniques work in different ways. If you wanted to visualize a protein in a living cell, which of these two imaging techniques would you use? How do you attach the fluorescent tag to your protein of choice?
GFP is used to label proteins using a fluorescent tag. You would genetically engineer your protein of choice to have the sequence for GFP and introduce that engineered gene in your cell. Then, when the protein is polymerized and GFP is expressed, you will be able to see the tag at the appropriate wavelength of light. Unlike GFP, immunofluorescence uses tagged antibodies (which have already been exposed to the appropriate antigens) to identify specific proteins. In this imaging technique, the cell has to be fixed, so you cannot look at living cells.
While scouring through the lab refrigerator for lunch, you find a suspicious fuzz growing in on your sandwich next to a refrigerated bottle of liquid penicillin. You wonder if the bacteria growing on your sandwich would be affected by penicillin or other antibiotics. Instead of tossing the sandwich, you take samples of the mysterious fuzz and prepare Gram-stained slides. a) What bacteria are more resistant to antibiotics? Why?
Gram negative bacteria have an outer membrane that is composed of lipopolysaccharides that confer some resistance to antibiotics and detergents.
c) Penicillin kills bacteria by preventing peptidoglycan crosslinking during cell division, which causes growing cells to bulge and ultimately burst. Assuming this bacteria is resistant to penicillin, what are examples of other cellular processes that could be targeted by antibiotics that this bacteria would be susceptible to?
Gram-negative bacteria are resistant to penicillin due to the lipopolysaccharides on their outer membrane. Therefore, they would not be susceptible to antibiotics that target similar processes. However, antibiotics that target cellular functions such as DNA replication, transcription, translation would be effective.
How do we know that the genes that allow yeast to divide or very similar to the ones that allow human cells to divide?
Hartwell believed that he could understand the process of cell division by introducing mutations randomly into a culture of yeast. He predicted that if you crippled the gene that was required to organize the process of cell division, this WHOLE cell would die. So he introduced point mutations randomly (single amino acid change) and then just isolated as many different colonies of temperature sensitive yeast as he could find and he saw some mutants that display after prolonged incubation at the higher temperature. There were different responses to different cells, some grew but didn't divide, others didn't grow. Hartwell and subsequently scientist Paul Nurse, using a different yeast, the two teams were able to assemble a pathway of dozens of genes that they could order in sequence to show which gene controlled which step in the cell division cycle. And then they demonstrated that these genes in yeast were conserved throughout the eukaryotic world, including even to the point where the same genes that yeast used to control cell division used in ourselves.
What are some other vacuole functions?
In plants and fungi, some carry out enzymatic hydrolysis (like lysosome in animal cells) In plants, small vacuoles can hold reserves of important organic compounds and help protect plant against herbivores by storing poisons
Where do ribosomes build proteins?
In two cytoplasmic regions: 1) free ribosomes, suspended in cytosol, mostly make cytosol proteins like sugar breakdown enzymes 2) bound ribosomes, attached to ER or nuclear envelope, generally make proteins destined for insertion into membranes for either packaging w/in lysosomes or secretion form cell like digestive enzymes secreted from the pancreas Both: structurally identical
How do electron microscopes work?
Instead of focusing light, they focus a beam of electrons through the specimen or onto its surface.
What are pore complexes?
Intricate protein structures that line each pore and regulate the entry and exit of proteins, RNAs, and larger macromolecules.
How is resolution related to wavelength?
Inversely: EM can produce much higher resolution because electron beams have much shorter wavelengths than visible light.
What is peptidoglycan layer in both gram positive/negative bacterium? How does it relate to penicillin?
It grows and shapes the bacterium and as the cell divides by efficient mechanism, it grows inward to help pinch the cell in half. The antibiotic penicillin acts by blocking an enzyme that's required to cross-link the bacterium at the point of division. And when that antibiotic is present, the cross-links don't form and because the bacterium is under high internal osmotic pressure, the cell wall begins to bulge at the point where these cross-links had been made. And then with time, the bacteria literally explodes because the internal osmotic pressure is so great that it ruptures the plasma membrane and the envelope falls apart. So when you take a drug, penicillin, you rely on the fact that the bacterium is actively growing. If it stops growing for whatever reason, it is no longer adding these cross-links and no longer susceptible to penicillin. So an actively growing infection of a gram-negative or gram positive bacterium is required actually for the effectiveness of penicillin. Now, you also know that as a result of microbial evolution. Bugs have developed a resistance to penicillin, and this is a common problem with virtually all antibiotics. There's an enzyme that simply chews up, degrades penicillin, penicillinace. And the gene that has evolved that enzyme is encoded on a small DNA molecule in a bacterial cell that is transmitted from cell to cell during division separately and can be transmitted by cells that make contact with each other through structures, through mating structures bacteria can make. So if you have one bacterium that harbors a plasmid, that houses the gene for penicillinace That bacterium can transmit that plasmid through a whole culture of bacteria very rapidly. Thus, the prevalence and rapid dissemination of antibiotic resistance encoded by genes on plasmids is a very serious problem. It is one of the great, most difficult problems and controlling bacterial infections. Infectious disease really continues to be a forefront issue.
In animal cells, what is the nucleus, what is it made up of, and what does it do?
It has three parts: 1) Nuclear envelope: double membrane enclosing the nucleus; perforated by pores and lined by the nuclear lamina; continuous w/ ER 2) Nucleolus: nonmembranous structure made up of densely stained granules and fibers adjoining parts of the chromatin. It is where ribosomal RNA is synthesized from genes in DNA. Also, in the nucleolus, proteins imported from the cytoplasm are assembled w/ rRNA into subunits of ribosomes, which then exit the nucleus to be assembled into ribosomes; a nucleus has one or more nucleoli. 3) Chromatin: material consisting of DNA and proteins; visible in a dividing cell as individual condensed chromosomes
What is the structure of the gram negative bacteria?
It has two membranes, a thinner peptidoglycan layer (inner membrane) and a very thick rigid outer membrane with surface structures called lipopolysaccharide.
What does the golgi stack do before dispatching its products from the trans face?
It sorts its products and targets them for their final destination by placing identifying tags (phosphate groups) on them which act like zip codes on mailing labels.
How does fluorescent microscopy work? (LM)
Locations of specific molecules are revealed by labeling the molecules w/ fluorescent dyes or antibodies, which absorb uv radiation and emit visible light. In this fluorescently labeled uterine cell, DNA is blue, mitochondria are orange, and the cell's cytoskeleton is green. These cells had to be permeable to the antibodies, and so you could only look at dead cells.
What are the functions of the rough ER?
Many cells secrete proteins produced by ribosomes attached to rough ER like pancreatic production of insulin protein. Polypeptide chain grows from a bound ribosome and then threaded into the ER lumen through a pore. New polypeptide folds into its functional shape as it enters the ER lumen. Most secretory proteins are glycoproteins (proteins w/ carbohydrates covalently bonded to them). Once the secretory proteins are formed, the ER membrane keeps them separate from proteins in the cytosol. The proteins then depart from the transitional ER wrapped in vesicle membranes. These transport vesicles move around the cell. Rough ER is also a Membrane factory for the cell, growing in place by adding membrane proteins and phospholipids to its own membrane. Similar to smooth ER, rough ER also makes membrane phospholipids, and as the ER membrane expands, portions of it are transferred as transport vesicles to other parts of the cell.
In animal cells, what is the microvilli, what is it made up of, and what does it do?
Membrane projections that increase the cell's surface area
Is archaea prokaryotic or eukaryotic?
Neither...it's its own thing
In animal cells, what is the endoplasmic reticulum (ER), what is it made up of, and what does it do?
Network of membranous sacs (cisternae) and tubes; active in membrane synthesis and other synthetic and metabolic processes; has rough (ribosome studded) and smooth regions. Transport vesicles bud off from the transitional ER on the rough ER and travel to the Golgi apparatus The ER membrane separates the internal compartment of the ER (lumen) or cisternal space from the cytosol. It's continuous w/ the nuclear envelope
In plant cells, what are the parts of the nucleus?
Nuclear envelope Nucleolus Chromatin
What is the nuclear lamina?
On the nucleus where the pores are not, the nuclear lamina runs as a netlike array of protein filaments that maintain the shape of the nucleus by mechanically supporting the nuclear envelope. May help organize genetic material
In animal cells, what is the golgi apparatus, what is it made up of, and what does it do?
Organelle active in synthesis, modification, sorting, and secretion of cell products
In animal cells, what is the peroxisome, what is it made up of, and what does it do?
Organelle w/ various specialized metabolic functions; produces H peroxide as a by-product and then converts it to H2O to detoxify alcohol
In animal cells, what is the mitochondrion, what is it made up of, and what does it do?
Organelle where cellular respiration occurs and most ATP is generated
In plant cells, what is the cell wall, what is it made up of, and what does it do?
Outer layer that maintains cell's shape and protects cell from mechanical damage; made of cellulose, other polysaccharides, and protein
Which types of life are made up of prokaryotic cells and which are made up of eukaryotic?
P: Bacteria, archaea E: Protists (diverse group of unicellular eukaryotes), fungi, animals, and plants
Explain how we were able to visualize cell structures:
Palade took an intense electron beam that projects down a vacuum chamber in which a very thin slice of tissue or cells is held on a grid. Though the electron microscope was invented in the early part of the 20th century in Germany, it was not suitable to be used for the inspection of living tissues because the damage to these tissues by an intense electron beam would = vaporize them. Palade perfected techniques to preserve tissue specimens. They found that by taking cells or tissues and dehydrating them, embedding them in a plastic resin after a chemical fixation, they could cut literally wavelength thin sections with a very sharp diamond knife. These specimens could then be held in a chamber that electrons were bombarded into, such that dense particles would reflect the electrons and clear less dense spaces. This would allow electrons to go through and the image, the shadow of the cell would be seen on a screen held beneath the vacuum chamber.
In plant cells, what is the chloroplast, what is it made up of, and what does it do?
Photosynthetic organelle; converts energy of sunlight to chemical energy stored in sugar molecules Double membraned
What happens in the Golgi apparatus?
Products of the ER (like proteins) are modified and stored and then sent to other destinations. Some macromolecule manufacturing occurs too such as pectins and noncellulose polysaccharides in plants. These nonprotein Golgi products are secreted from the trans face inside transport vesicles and fuse w/ the plasma membrane, and then are released.
In animal cells, what is the cytoskeleton, what is it made up of, and what does it do?
Reinforces cell's shape; functions in cell movement; components are made of protein. Includes microfilaments, intermediate filaments, and microtubules
In animal cells, what is the centrosome, what is it made up of, and what does it do?
Region where the cell's microtubules are initiated; contains a pair of centrioles
How were spectral variants of GFP made?
Roger Chen recognized that the intrinsic fluorescence of this molecule might be changed by introducing mutations into residues surrounding the tryptophan that might alter the fluorescence absorption and emission spectrum. by introducing mutations into the gene for GFP, the GFP spectrum would be shifted and you'd create variants of GFP that emit light at different wavelengths. So, you can tag almost a dozen different proteins in the same cell and you can then see different molecules and structures moving around w/ respect to each other in a living cell.
You are running the multi-subunit E. coli RNA Polymerase (RNAP) on a polyacrylamide gel in the presence of SDS. What is the role of SDS? What is the role of polyacrylamide?
SDS is a detergent that completely disrupts secondary and tertiary structure. It also masks the polypeptides charge and coats it with a negative charge that allows it to migrate towards the positive pole (anode in electrophoresis). Polyacrylamide forms a mesh gel matrix through which the proteins travel, with larger proteins taking longer to go through the matrix than smaller proteins
How do you isolate proteins in order to study them?
Scientific team found that they could fractionate the pancreas by grinding the tissue open, obtaining a soluble fraction from pancreatic extract, which was happened to be a partially pure sample of this protein insulin, initially isolated from the pancreas of a sacrificed dog. The partially purified insulin from dog pancreas was used in the early 1920's to treat diabetic patients. This process began by homogenizing (blending) a tissue, by breaking it down to obtain a soluble protein. And after having sedimented the large particles, the supernatant fraction, can be analyzed based off of size, shape, and charge.
What is the experimental goal of SDS-PAGE?
Separating a protein of a known size
b) What color would you expect this bacteria to be, if it were more resistant to antibiotics? Why?
Since gram-negative bacteria are more resistant to antibiotics, we would expect our gram-stained bacteria to be red. Since the peptidoglycan layer is covered by the outer membrane, the crystal violet is rinsed away, revealing red safrinin dye.
How does cryo-electron microscopy work? cryo-SEM) Why is it used?
Specimens of tissue or aq solutions of proteins are frozen rapidly at temps less than -160C, locking the molecules into a rigid state. A beam of electrons is passed thru the sample to visualize the molecules by electron microscopy, and software is used to merge a series of such micrographs, creating a 3-D image. It is used to preserve living cells at low temperatures, which avoids the use of preservatives, allowing visualization of structures in their cellular environment.
What are the functions of the smooth ER?
Synthesizes lipids (like sex hormones and steroid hormones secreted by the adrenal gland) Metabolizes carbohydrates Detoxifies drugs and poisons (by adding hydroxyl groups to drug molecules, making them more water soluble and flushable) Stores calcium ions (in muscle cells, the smooth ER membrane pumps calcium ions from cytosol into the ER lumen, and when muscle is stimulated by a nerve impulse, calcium ions rush back across the ER membrane into the cytosol and trigger contraction of the muscle cell)
What does the golgi apparatus consist of?
The Golgi apparatus consists of flattened membranous sacs—cisternae (not connected like they are in the ER)—looking like a stack of pita bread. Vesicles in the golgi apparatus transfer material between parts of the Golgi and other structures.
Where do transport vesicles go after leaving the ER?
The Golgi apparatus, which is a warehouse for receiving, sorting, shipping, and some manufacturing.
What is autophagy?
The cell recycles its own organic material. Lysosome will surround it and eat it, allowing its building blocks to be something else. First, lysosomes fuse w/ vesicle containing damaged organelles. Second, lysosomal enzymes dismantle the inner membrane and the enclosed material, and the resulting small organic compounds are released to the cytosol for reuse.
What are contractile vacuoles?
found in many freshwater protists, pump excess water out of cells
What does the cisternal maturation model state?
The cisternae of the Golgi progress forward from the cis to the trans face, carrying and modifying their cargo as they move. This is a rejection of the early belief that Golgi was a static structure w/ products in various stages of processing transferred from one cisterna to the next by vesicles.
How does confocal microscopy work? (LM)
The image shows two types of fluorescence micrographs: confocal (top) and standard (bottom). (Nerve cells are green, support cells=orange, areas of overlap=yellow). In confocal microscopy, a laser is used to create a single plane of fluorescence; out of foucs light form other planes is eliminated. by capturing sharp images at many different planes, a 3-D reconstruction can be created. A standard fluorescence micrograph is blurry cuz out of focus light is not excluded.
What are the drawbacks of inherited lysosomal storage disease?
The lack of a functioning hydrolytic enzyme normally present in lysosomes makes the lysosome engorged w/ indigestible material. In Tay Sachs disease, the lipid-digesting enzyme is missing and the brain becomes impaired by an accumulation of lipids in cells
What is a major disadvantage to using EM even if it is more powerful than LM?
The methods used to prepare the specimen in EM kill the cells and can introduce structural features seen in micrographs that don't exist in living cells called artifacts.
What is cell fractionation?
The process where cells are broken up and the different organelles they contain are separated out. This process is conducted w/ a centrifuge, which spins test tubes holding mixtures of disrupted cells at a series of increasing speeds (differential centrifugation). At each speed, the resulting force causes a subset of the cell components to settle at bottom of tube, forming a pellet with larger components at lower speeds and smaller components at higher speeds.
How does deconvolution microscopy work? (LM)
The top of this image of a white blood cell was reconstructed from many blurry fluorescence images at different planes, each processed using deconvolution software. This process digitally removes out of focus light and reassigns it to its source, creating a much sharper 3D image. The bottom is a compilation of standard fluorescent micrographs through the same cell.
What is the structure of the gram positive bacteria? How can they be distinguished?
They don't really have an outer membrane, unlike gram negative, it has a very thick peptidoglycan layer that surrounds the cell and gives its structural rigidity, protecting the inner membrane of the bacteria. These types of bacteria can be distinguished by applying a crystal violet stain. And when the stain is mixed with gram positive bacteria, the stain is picked up quite vividly by the peptidoglycan layer, giving the bacteremic a very distinct appearance from that gram-negative bacteria. (cell wall is sufficiently thick so that it traps the purple dye to mask the red dye
How does scanning electron microscopy work? (SEM)
They're especially useful for studying the topography of a specimen. The electron beam scans the surface of the sample, and the beam excites electrons on the surface, which are detected by a device that translates the pattern of electrons into an electronic signal sent to a video screen, creating a 3D looking image.
In animal cells, what is the ribosomes, what is it made up of, and what does it do?
They're made of rRNA and proteins. They're complexes that make proteins; free in cytosol or bound to rough ER or nuclear envelope. Not considered organelles cuz not membrane bound
How are endomembrane organelles connected?
Through direct physical contact or by the transfer of membrane segments as tiny vesicles (sacs of membrane)
What has X-ray crystallography been used for?
To determine the atomic structure of many large molecules In this approach, a regular, highly arranged crystal of a protein can be exposed to an x-ray beam. And if the crystal is stable to this beam, the x-rays bend in reflecting from atomic positions. The pattern of this diffraction of x-rays is registered on a plate. And the position of the diffraction spectrum can be computationally deconvoluted into an atomic structure of a protein.
How does super resolution microscopy work? (LM)
To make this super resolution image of a cow aorta cell (top), individual fluorescent molecules were excited by UV light and their position recorded. (DNA is blue, mitochondria red, and part of the cytoskeleton green). Combining info from many molecules in different places "breaks" the resolution limit, resulting in the sharp image on top. Tag individual molecules in cells
How does Transmission electron microscopy work? (TEM)
Used to study internal structure of cells. The TEM aims an electron beam through a very thin section of the specimen. For the TEM, the specimen has been stained w/ atoms of heavy metals, which attach to certain cellular structures, thus enhancing the electron density of some parts of the cell more than others. The electrons passing through the specimen are scattered more in the denser regions, so fewer are scattered more in the denser regions, so fewer are transmitted.
How does phase-contrast microscopy work? (LM)
Variations in density w/in the specimen are amplified to enhance contrast in unstained cells; especially useful for examining living, unstained cells.
3. You are a biochemist and have just purified a human protein with a novel catalytic activity. You wish to find out exactly how the substrate for this enzyme binds to the catalytic site. Which one of the following techniques would you use? a. Mix the protein with or without the substrate and evaluate on an SDS polyacrylamide gel to determine if the substrate changes the electrophoretic mobility of the protein. b. Clone the gene for the protein and fuse it to the gene for GFP and use fluorescence microscopy to examine cells grown with or without the substrate. c. Form crystals of the protein in the presence or absence of substrate and examine the structures of the two by X-ray crystallography. d. Examine the gene that encodes this enzyme from a variety of organisms to detect amino acid changes that are the most highly variable.
c. Form crystals of the protein in the presence or absence of substrate and examine the structures of the two by X-ray crystallography.
You are an eager young investigator starting a research project using genetics to discover the genes required for eukaryotic cellular DNA replication. Which one of the following techniques and applications would be the best place to start? a. Look for gene deletion mutations that block DNA replication in human cells grown in cell culture. b. Look for mutations in yeast cells that block DNA replication but not colony formation at 37C. c. Look for mutations in E. coli cells that block DNA replication and colony formation at 42C. d. Look for mutations in yeast cells that block DNA replication and colony formation at 37C.
d. Look for mutations in yeast cells that block DNA replication and colony formation at 37C.
What are the three parameters in microscopy?
magnification: ratio of an object's image size to real size resolution: the clarity of the image, min distance two points can be separated and still be distinguished contrast: difference in brightness between light and dark areas
In plant cells, what is the cytoskeleton made up of?
microfilaments Microtubules
In animal cells, what is the flagellum, what is it made up of, and what does it do?
moving structure present in some animal cells, composed of a cluster of microtubules w/in an extension of the plasma membrane
What is a central vacuole?
only in plants, store water and develop by the merging of smaller vacuoles. Solution inside a central vacuole is called cell sap and is the plant's main repository of inorganic ions (K/Cl) As it holds more water, plant can grow bigger
What happens in a light microscope?
visible light is passed through the specimen and then through glass lenses. The lenses refract (bend) the light in such a way that the image of the specimen is magnified as it is projected into the eye or into a camera.
