PCB 3023 Exam 3

DNA library

Collection of cloned DNA molecules, representing either an entire genome (genomic library) or copies of the mRNA produced by a cell (cDNA library). (page 339)

phosphatidylcholine

Common phospholipid present in abundance in most cell membranes; uses choline attached to a phosphate as its head group. (page 367)

secretion

Production and release of a substance from a cell. (page 522)

DNA cloning

Production of many identical copies of a DNA sequence. (page 334)

glycocalyx

Protective layer of carbohydrates on the outside surface of the plasma membrane formed by the sugar residues of membrane glycoproteins, proteoglycans, and glycolipids. (page 383)

clathrin

Protein that makes up the coat of a type of transport vesicle that buds from either the Golgi apparatus (on the outward secretory pathway) or from the plasma membrane (on the inward endocytic pathway). (page 512)

CRISPR

System for gene editing based on a bacterial enzyme that uses a guide RNA molecule to search for and modify specific nucleotide sequences in the genome. (page 358)

detergent

Soapy substance used to solubilize lipids and membrane proteins. (page 378)

tethering protein

Filamentous transmembrane protein involved in the docking of transport vesicles to target membranes. (page 514)

fat droplet

Large cluster of hydrophobic fats or oils that forms inside the cells. (page 369)

unsaturated

Describes an organic molecule that contains one or more double or triple bonds between its carbon atoms. (page 371)

Watch the animation on mitochondrial protein import, and then answer the questions. Put the following steps used to transport proteins into mitochondria into the proper order.

1. The mitochondrial protein is synthesized in the cytosol 2. The receptor on the mitochondrial membrane binds the signal sequence on the protein 3. The protein is delivered to the translocarion apparatus on the mitochondria 4. The protein is passed through the translocation apparatus 5. The signal sequence is removed by signal peptidase and the protein folds into its finals FEEDBACK: Once protein synthesis is complete in the cytosol, a receptor on the mitochondrial membrane binds the signal sequence found at the N-terminus of the protein. The receptor delivers the protein to the translocation apparatus that spans both the inner and outer mitochondrial membranes and the unfolded protein is passed through the translocation apparatus. Chaperone proteins inside the mitochondria help pull the protein into the mitochondria. Once transport is complete, signal peptidase removes the signal sequence and the protein folds into its final conformation.

How thick is the plasma membrane?

50 atoms FEEDBACK: Correct. The plasma membrane is so thin it cannot be seen directly even with a light microscope.

Approximately what percentage of the volume of a typical eukaryotic cell is comprised of cytosol?

50% FEEDBACK: The volume of a typical eukaryotic cell is about 50% cytosol and the remaining 50% is occupied by membrane-bound organelles.

recombinant DNA

A DNA molecule that is composed of DNA sequences from different sources. (page 337)

phagocytic cell

A cell such as a macrophage or neutrophil that is specialized to take up particles and microorganisms by phagocytosis. (page 523)

gene knockout

A genetically engineered animal in which a specific gene has been inactivated. (page 356)

phospholipid

A major type of lipid molecule in many cell membranes. Generally composed of two fatty acid tails linked to one of a variety of phosphate-containing polar groups. (page 367)

transgenic organism

A plant or animal that has stably incorporated into its genome one or more genes derived from another cell or organism. (page 355)

membrane protein

A protein associated with the lipid bilayer of a cell membrane. (page 375)

RNA interference (RNAi)

Cellular mechanism activated by double-stranded RNA molecules that results in the destruction of RNAs containing a similar nucleotide sequence. It is widely exploited as an experimental tool for preventing the expression of selected genes (gene silencing). (page 355)

nuclear pore

Channel through which selected large molecules move between the nucleus and the cytoplasm. (page 503)

Watch the animation about the unfolded protein response, and then answer the questions. Three separate pathways make up the unfolded protein response in the ER. Sort the following characteristics of the unfolded protein responses into the correct pathway.

All Three: -Activates transcription of specific genes -Increases the ability of the ER to fold more proteins IRE1 Pathway: -Leads to removal of an intron from a specific RNA and translation of the mRNA -Contains both kinase and RNAse domains PERK Pathway: -Phosphorylates a translation initiation factor, leading to a global reduction in translation ATF6 Pathway: -Requires cleavage of the protein -Travels the Golgi apparatus and the nucleus before activating transcription FEEDBACK: Each of the three pathways activates transcription of specific genes that increase the ability of the ER to fold more proteins. The IRE1 pathway starts with a protein with both kinase and RNAse domains that, when activated, removes an intron from a specific RNA and translation of the protein. The PERK pathway also starts with a kinase but in this case phosphorylates a translation initiation factor, reducing global translation. Finally, the ATF6 pathway does not have a kinase, but instead activation of the pathway causes ATF6 to travel to the Golgi, where it is cleaved before transport to the nucleus, where it activates transcription of specific genes.

signal sequence

Amino acid sequence that directs a protein to a specific location in the cell, such as the nucleus or mitochondria. (page 502)

Which of the following is true of lysosomes?

An ATP-driven H+ pump in the lysosomal membrane maintains the organelle's pH. FEEDBACK: Correct. The lysosome is maintained at an acidic pH by a pump in its membrane that hydrolyzes ATP to transport H+ into the lumen.

membrane-enclosed organelle

Any organelle in a eukaryotic cell that is surrounded by a lipid bilayer—for example, the endoplasmic reticulum, Golgi apparatus, and lysosome. (page 496)

cDNA library

Collection of DNA fragments synthesized using all of the mRNAs present in a particular type of cell as a template. (page 339)

Identify each enzyme step by dragging the enzyme label to its correct target. There are four labels and only two targets, so two labels are not used.

B, C FEEDBACK: Correct. To insert a piece of DNA into a plasmid vector, the purified plasmid DNA is opened up by a restriction enzyme that cleaves it at a single site, and the DNA fragment to be cloned is then spliced into that site using DNA ligase.

Which of the following accurately describes a step in GTP-driven nuclear transport?

Binding of Ran-GTP to the receptor releases the cargo protein. FEEDBACK: GTP hydrolysis ensures that nuclear import occurs in the proper direction. This is accomplished by a small GTPase protein called Ran. In the nucleus it is in its GTP-bound form and binds to nuclear import receptors, causing them to release their cargo. It is then shuttled back to the cytosol with the nuclear import receptor, and upon entry, hydrolysis of GTP is stimulated, and leads to release of the receptor to bind more cargo.

Watch the animation on receptor-mediated endocytosis, and then answer the questions. Many viruses enter cells through receptor-mediated endocytosis. Which of the following strategies could be affective in blocking entry of this class of viruses into cells and could be used to treat viral infections?

Block the function of adaptin. Block the receptor with an antibody. FEEDBACK: Some viruses such as the human rhinovirus 2 gain entry into their host cells through receptor-mediated endocytosis by binding the LDL receptor on the surface of cells. Developing drugs that could block the entry of substrates into the cell by receptor-mediated endocytosis might be an effective treatment for things like colds caused by the rhinovirus or to treat other infections caused by viruses that enter the cell through receptor-mediated endocytosis. The receptor, adaptin, and clathrin are all required for viral entry so inhibition of any of these three would block receptor-mediated endocytosis. Increasing clathrin activity would not prevent viral entry. Actin filaments are not involved in receptor-mediated endocytosis, so blocking them would not affect viral entry.

How do clathrin-coated vesicles select their cargo molecules?

Cargo receptors bind specifically to cargo proteins and to clathrin. FEEDBACK: Vesicles destined for different compartments have different types of protein coats. The cargo for these vesicles is selected by specifically binding to cargo receptors that interact with a specific type of protein coat.

Watch the animation about clathrin, and then answer the questions. Match the following structures used in receptor-mediated endocytosis with their functions.

Cargo: Molecules packaged into vesicle for transport Receptor: Captures the correct cargo molecules Adaptin: Mediates contact between the receptor and another component Clathrin: Shapes the forming vesicle FEEDBACK: Receptor-mediated endocytosis occurs when cargo molecules to be imported into the cell are bound by a specific receptor in the plasma membrane. An adaptin protein binds to the receptor and acts as an adaptor or bridge between the receptor bound to cargo and the clathrin molecule. The growing vesicle is shaped by the clathrin molecules, which form a closed cage around the vesicle. After formation, the vesicle is uncoated and clathrin leaves. The vesicle is then transported to the proper intracellular location.

genomic library

Collection of cloned DNA molecules that represents the entire genome of a cell. (page 339)

complementary DNA (cDNA)

DNA molecule synthesized from an mRNA molecule and therefore lacking the introns that are present in genomic DNA. (page 339)

saturated

Describes an organic molecule that contains a full complement of hydrogen; in other words, no double or triple carbon-carbon bonds. (page 372)

Researchers studying yeast discovered that, for some mutants, when the temperature at which the cells are grown is elevated from 25ºC to 37ºC, their secretory pathway no longer functions and the cells grow dense with unsecreted protein. When these cells are examined microscopically, they can be divided into groups that vary in terms of where the unsecreted proteins accumulate. In some of the mutants, proteins accumulate in the ER; in others, the Golgi; in others, they accumulate in vesicles near the plasma membrane.What is the likely explanation for this difference in appearance?

Different temperature-sensitive mutations affect different stages of the transport process. FEEDBACK: Movement of proteins between different cell compartments via transport vesicles has been studied extensively using genetic techniques. Studies of mutant yeast cells that are defective for secretion at high temperatures have identified numerous genes involved in carrying proteins from the ER to the cell surface. Many of these mutant genes encode temperature-sensitive proteins. These mutant proteins may function normally at 25°C, but when the yeast cells are shifted to 37°C, the proteins are inactivated. As a result, when researchers raise the temperature, the various proteins destined for secretion instead accumulate inappropriately in the ER, Golgi apparatus, or transport vesicles—depending on the particular mutation.Using this approach, one research group identified at least 23 different genes required for the transport of proteins from their site of synthesis to their secretion at the cell surface.

nuclear envelope

Double membrane surrounding the nucleus. Consists of outer and inner membranes, perforated by nuclear pores. (page 503)

A transmembrane protein has the structure shown. If an ER signal sequence were added to its N-terminus, which structure would the engineered protein adopt?

E FEEDBACK: In the original protein, an internal (rather than an N-terminal) signal sequence (numbered 1) is used to start the protein transfer; this internal signal sequence, called a start-transfer sequence, is never removed from the polypeptide but remains a transmembrane helix. This arrangement occurs in some transmembrane proteins in which the polypeptide chain passes back and forth across the lipid bilayer. In such cases, the internal start-transfer sequence serves to initiate translocation, which continues until a stop-transfer sequence (numbered 2) is reached.If a signal sequence were added to the protein's N-terminus (gray), this new sequence would initiate translocation. The polypeptide chain that follows this sequence would therefore be located in the ER lumen.The next hydrophobic sequence (numbered 1) would then halt translocation, and the one that follows would re-initiate translocation. This alteration would effectively turn the protein upside down; the N-terminal signal sequence would presumably be removed by a signal peptidase whose active site faces the lumenal side of the ER membrane.

restriction enzyme

Enzyme that can cleave a DNA molecule at a specific, short sequence of nucleotides. Extensively used in recombinant DNA technology. (page 335)

DNA ligase

Enzyme that seals nicks that arise in the backbone of a DNA molecule; in the laboratory, can be used to join together two DNA fragments. (page 337)

Which of the following is a difference between exocytic and endocytic pathways?

Exocytic pathways often start with synthesis of proteins, whereas endocytic pathways involve breaking down macromolecules like proteins. FEEDBACK: Endocytic pathways and exocytic pathways both use transport vesicles to move lipids, membrane components, proteins, and soluble molecules from the outside of the cell to inside of the cell or vice versa. Endocytic pathways bring molecules in from the outside of the cell into an endosome, which can then mature into a lysosome. Endocytosed molecules do not travel to the Golgi in vesicles.

hybridization

Experimental technique in which two complementary nucleic acid strands come together and form hydrogen bonds to produce a double helix; used to detect specific nucleotide sequences in either DNA or RNA. (page 341)

green fluorescent protein (GFP)

Fluorescent protein, isolated from a jellyfish, that is used experimentally as a marker for monitoring the location and movement of proteins in living cells. (page 353)

cell cortex

Specialized layer of cytoplasm on the inner face of the plasma membrane. In animal cells, it is rich in actin filaments that govern cell shape and drive cell movement. (page 380)

membrane domain

Functionally and structurally specialized region in the membrane of a cell or organelle; typically characterized by the presence of specific proteins. (page 381)

reporter gene

Gene encoding a protein whose activity is easy to monitor experimentally; used to study the expression pattern of a target gene or the localization of its protein product. (page 352)

Which of the following organelles is surrounded by a single membrane?

Golgi apparatus FEEDBACK: The nucleus, mitochondria, and chloroplasts are each surrounded by a double membrane, and organelles like the Golgi apparatus, lysosomes, and the endoplasmic reticulum have a single membrane.

amphipathic

Having both hydrophobic and hydrophilic regions, as in a phospholipid or a detergent molecule. (page 367)

Watch the animation about the unfolded protein response, and then answer the questions. You complete a further experiment by treating your cells with an RNAse inhibitor and get the results shown in Figure B. Given the results of Figure A and Figure B, what pathway(s) is/are important for this cell line?

IRE1 FEEDBACK: IRE1 and PERK both have protein kinase activity, but only IRE1 is also an RNAse. Since blocking RNAse activity also inhibits the unfolded protein response, IRE1 must be the major pathway in these cells. The two experiments together allow you to narrow down the response in these cells to the IRE1 pathway.

Watch the animation about the unfolded protein response, and then answer the questions. The three pathways of the unfolded protein response differ in importance in different cell types, enabling cells to tailor the response to their individual needs. You join a lab that studies the relative importance of the UPR in different cell types. Your advisor gives you a new cell culture and directs you to determine which of the three pathways is the most important for that cell type. You first treat the cells with a kinase inhibitor. Given the results in Figure A, which pathway(s) might be important in these cells?

IRE1 and PERK FEEDBACK: IRE1 and PERK are both protein kinases that phosphorylate their dimerization partner after binding. In both cases, phosphorylation of the kinase further activates the activity of IRE1 and PERK and leads to the downstream signaling events and activation of the unfolded protein response. Blocking phosphorylation would inhibit activation of the unfolded protein response by these two proteins. This experiment is not enough to determine whether IRE1 or PERK is more important in this particular cell type.

endomembrane system

Interconnected network of membrane-enclosed organelles in a eukaryotic cell; includes the endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, and endosomes. (page 499)

Which statements are true of receptor-mediated endocytosis?

It allows the internalization of extracellular substances in clathrin-coated vesicles. The process can be hijacked by viruses to gain entry into cells. It allows cholesterol-carrying low-density lipoproteins (LDLs) to be taken up by cells. FEEDBACK: In most animal cells, specific macromolecules can be taken up from the extracellular fluid via clathrin-coated vesicles. The macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles. This process, called receptor-mediated endocytosis, provides a selective concentrating mechanism that increases the efficiency of internalization of particular macromolecules more than 1000-fold compared with ordinary pinocytosis. Such is the case when animal cells import the cholesterol they need to make new membranes.Cholesterol is transported in the bloodstream bound to proteins in the form of particles called low-density lipoproteins, or LDL. Cholesterol-containing LDLs bound to receptors on the cell surface are ingested by receptor-mediated endocytosis and delivered to endosomes. In the acidic interior of endosomes, the LDL dissociates from its receptor. These empty receptors are returned, via transport vesicles, to the plasma membrane for reuse. The LDL is delivered to lysosomes, where it is broken down, allowing free cholesterol to enter the cytosol.Receptor-mediated endocytosis is also used to import many other essential metabolites: the vitamin B12 and iron required to make hemoglobin is taken up by immature red blood cells. At the same time, receptor-mediated endocytosis can also be exploited by viruses, such as the influenza virus, to gain entry into cells.

endoplasmic reticulum (ER)

Labyrinthine membrane-enclosed compartment in the cytoplasm of eukaryotic cells where lipids and proteins are made. (page 507)

autophagy

Mechanism by which a cell "eats itself," digesting molecules and organelles that are damaged or obsolete. (page 528)

receptor-mediated endocytosis

Mechanism of selective uptake of material by animal cells in which a macromolecule binds to a receptor in the plasma membrane and enters the cell in a clathrin-coated vesicle. (page 525)

transport vesicle

Membrane vesicle that carries proteins from one intracellular compartment to another—for example, from the endoplasmic reticulum to the Golgi apparatus. (page 511)

endosome

Membrane-enclosed compartment of a eukaryotic cell through which material ingested by endocytosis passes on its way to lysosomes. (page 526)

Golgi apparatus

Membrane-enclosed organelle in eukaryotic cells that modifies the proteins and lipids made in the endoplasmic reticulum and sorts them for transport to other sites. (page 518)

secretory vesicle

Membrane-enclosed organelle in which molecules destined for secretion are stored prior to release. (page 522)

lysosome

Membrane-enclosed organelle that breaks down worn-out proteins and organelles and other waste materials, as well as molecules taken up by endocytosis; contains digestive enzymes that are typically most active at the acid pH found inside these organelles. (page 527)

unfolded protein response (UPR)

Molecular program triggered by the accumulation of misfolded proteins in the endoplasmic reticulum. Allows cells to expand the endoplasmic reticulum and produce more of the molecular machinery needed to restore proper protein folding and processing. (page 518)

vesicular transport

Movement of material between organelles in the eukaryotic cell via membrane-enclosed vesicles. (page 511)

How does the nuclear pore restrict the passage of large molecules that do not bear the correct nuclear localization signal?

Nuclear pore proteins contain disordered segments that form a gel-like meshwork inside the pore. FEEDBACK: Many of the proteins that line the nuclear pore contain extensive, unstructured regions in which the polypeptide chains are largely disordered. These disordered segments form a soft, tangled meshwork—like a kelp forest—that fills the center of the channel, preventing the passage of large molecules but allowing small, water-soluble molecules to pass freely and nonselectively between the nucleus and the cytosol.Nuclear import receptors, carrying proteins bound for the nucleus, can penetrate through this tangle by grabbing onto short, repeated amino acid sequences within the segments that fill the center of the pore. When the nuclear pore is empty, these repeated sequences bind to one another, forming a loosely packed gel. Nuclear import receptors interrupt these interactions, and thereby open a local passageway through the meshwork. The import receptors then bump along from one repeat sequence to the next, until they enter the nucleus and deliver their cargo.

What is true of protein glycosylation in the ER?

Oligosaccharides are added by an enzyme that has its active site on the lumenal side of the ER membrane. FEEDBACK: In the ER, individual sugars are not added one by one to the protein to create an oligosaccharide side chain. Instead, a preformed, branched oligosaccharide containing a total of 14 sugars is attached en bloc to all proteins that carry the appropriate site for glycosylation. The oligosaccharide is originally attached to a specialized lipid, called dolichol, in the ER membrane; it is then transferred to the amino (NH2) group of an asparagine side chain on the protein, immediately after a target asparagine emerges in the ER lumen during protein translocation. The addition takes place in a single enzymatic step that is catalyzed by a membrane-bound enzyme (an oligosaccharyl transferase) that has its active site exposed on the lumenal side of the ER membrane—which explains why cytosolic proteins are not glycosylated in this way.A simple sequence of three amino acids, of which the target asparagine is one, defines which sites in a protein receive the oligosaccharide. Oligosaccharide side chains linked to an asparagine NH2 group in a protein are said to be N-linked, and this is by far the most common type of linkage found on glycoproteins.

SNARE

One of a family of membrane proteins responsible for the selective fusion of vesicles with a target membrane inside the cell. (page 514)

Rab protein

One of a family of small GTP-binding proteins present on the surfaces of transport vesicles and organelles that serves as a molecular marker to help ensure that transport vesicles fuse only with the correct membrane. (page 514)

bacteriorhodopsin

Pigmented protein found in abundance in the plasma membrane of the salt-loving archaeon Halobacterium halobium; pumps protons out of the cell, fueled by light energy. (page 379)

endocytosis

Process by which cells take in materials through an invagination of the plasma membrane, which surrounds the ingested material in a membrane-enclosed vesicle. (See also pinocytosis and phagocytosis.) (page 523)

exocytosis

Process by which most molecules are secreted from a eukaryotic cell. These molecules are packaged in membrane-enclosed vesicles that fuse with the plasma membrane, releasing their contents to the outside. (page 515)

What is one of the main differences in the behavior of the proteins in a vesicle destined for constitutive secretion, and the proteins in the vesicle destined for regulated secretion?

Proteins in the regulated secretion vesicle tend to aggregate and become highly concentrated in the ionic conditions in the vesicle. FEEDBACK: Constitutive secretion vesicles contain lipids and proteins that are continuously supplying the plasma membrane with new components. Proteins in regulated secretion vesicles form concentrated aggregates so that when they are released in response to a signal, the levels of the protein can rapidly increase.

What can serve as the original template material for the polymerase chain reaction?

RNA & DNA FEEDBACK: Correct. Both DNA and RNA can serve as the original template material for the polymerase chain reaction. When the template is RNA, as it would be for detecting HIV in a sample of blood, the RNA is first converted to DNA by reverse transcriptase.

rough endoplasmic reticulum

Region of the endoplasmic reticulum associated with ribosomes and involved in the synthesis of secreted and membrane-bound proteins. (page 507)

RNA-Seq

Sequencing technique used to determine directly the nucleotide sequence of a collection of RNAs. (page 352)

cholesterol

Short, rigid lipid molecule present in large amounts in the plasma membranes of animal cells, where it makes the lipid bilayer less flexible. (page 372)

peroxisome

Small membrane-enclosed organelle that contains enzymes that degrade lipids and destroy toxins. (page 506)

coated vesicle

Small membrane-enclosed sac that wears a distinctive layer of proteins on its cytosolic surface. It is formed by pinching-off of a protein-coated region of cell membrane. (page 512)

Plasmid

Small, circular DNA molecule that replicates independently of the genome. Used extensively as a vector for DNA cloning. (page 337)

polymerase chain reaction (PCR)

Technique for amplifying selected regions of DNA by multiple cycles of DNA synthesis; can produce billions of copies of a given sequence in a matter of hours. (page 341)

in situ hybridization

Technique in which a single-stranded RNA or DNA probe is used to locate a complementary nucleotide sequence in a chromosome, cell, or tissue; used to diagnose genetic disorders or to track gene expression. (page 352)

Fluorescence recovery after photobleaching (FRAP) is used to monitor the movement of fluorescently labeled molecules within the plane of a cell membrane. The molecules labeled are often proteins, but lipids can be labeled too.How would the curve that represents FRAP for labeled proteins compare to the curve representing labeled lipids?

The FRAP curve for lipids would show a much more rapid recovery to initial levels of fluorescence. FEEDBACK: Because lipids move much faster than proteins, recovery of fluorescence for labeled lipids would be much more rapid and should reach the initial level of fluorescence fairly quickly.

Why do phospholipids form bilayers in water?

The hydrophilic head is attracted to water, while the hydrophobic tail shuns water. FEEDBACK: Correct. The hydrophilic head can form electrostatic attractions and hydrogen bonds with water, while the hydrophobic tails are insoluble in water.

phagocytosis

The process by which particulate material is engulfed ("eaten") by a cell. Prominent in predatory cells, such as Amoeba proteus, and in cells of the vertebrate immune system, such as macrophages. (See also endocytosis.) (page 523)

Watch the animation on mitochondrial protein import, and then answer the questions. ATP is important for chaperone protein function. Why would protein import into mitochondria be disrupted if ATP were depleted from inside mitochondria?

The protein could slip back out of the mitochondria during transport. FEEDBACK: The chaperones in mitochondria bind to the unfolded protein as it is inserted through the mitochondrial translocation apparatus. Binding of chaperones helps pull the protein into the mitochondria and prevents the unfolded protein from exiting back out. ATP is required for chaperone function, so a lack of ATP would inhibit the function of chaperones and would allow proteins to slip back out of the mitochondria.

Ricin is one of the most powerful toxins known. The protein consists of two subunits: the A chain is an enzyme that inhibits translation and the B chain is a lectin that binds to carbohydrates on the cell surface. What is the most likely mechanism by which ricin enters the cell?

The protein is internalized by endocytosis. FEEDBACK: Ricin is a powerful toxin produced by the castor bean plant. Less than 2 mg injected into the bloodstream will kill an adult human. The protein is a heterodimer composed of an A chain, which inhibits protein translation, and a B chain, which binds to carbohydrates and glycoproteins on the cell surface. Because ricin is a large protein, it must be taken into the cell via endocytosis. The toxin is internalized by both receptor-mediated endocytosis and pinocytosis. It is then transported via endosomes to the Golgi apparatus and from there into the ER. It escapes from the ER to the cytosol by partially unfolding in the ER lumen, triggering its release to the cytosol for degradation. Once in the cytosol, the A chain refolds and exerts its toxic influence on protein synthesis.

plasma membrane

The protein-containing lipid bilayer that surrounds a living cell. (page 365)

To determine whether a signal sequence directs proteins to a particular organelle, researchers prepare two versions of the same protein: one version contains the signal sequence, while the other lacks it. They label the protein that contains the signal sequence with a radioactive marker, and then incubate both of the proteins with the organelle of interest.After allowing enough time for any of the proteins to be transported into the organelle, a protease is added to the mixture.If the signal sequence is the correct one for the selected organelle, what would the researchers likely see?

The radioactive label would be associated with an intact protein. FEEDBACK: A protein bearing a signal sequence can be introduced to a preparation of isolated organelles in a test tube. This mixture can then be tested to see whether the protein is taken up by the organelle. The protein is usually produced in vitro by cell-free translation of a purified mRNA encoding the polypeptide; in the process, radioactive amino acids can be used to label the protein so that it is easy to isolate and to follow. The labeled protein is incubated with a selected organelle and its translocation is monitored by one of several methods.In one approach, the labeled protein can be incubated with the organelle and a protease can be added to the preparation. If the protein bearing the signal sequence is transported into the organelle, it will be selectively protected from digestion by the organelle membrane; adding a detergent that disrupts the organelle membrane will eliminate that protection, and the transported protein will also be degraded.

dideoxy (Sanger) sequencing

The standard method of determining the nucleotide sequence of DNA; utilizes DNA polymerase and a set of chain-terminating nucleotides. (page 346)

In a patch of animal cell membrane about 10 μm in area, which will be true?

There will be more lipids than proteins. FEEDBACK: Correct. Proteins constitute about half the mass of an animal cell membrane. Therefore, in terms of mass, proteins and lipids provide an equal share.However, lipids are much smaller than proteins, so a cell membrane typically contains 50 times more lipid molecules than protein molecules.

lipid bilayer

Thin pair of closely juxtaposed sheets, composed mainly of phospholipid molecules, that forms the structural basis for all cell membranes. (page 367)

Genetic engineering in plants is made simpler by what fact?

Transgenic plants can be grown from almost any type of plant cells transfected with DNA in culture. FEEDBACK: Correct. Cells can be cut from an adult plant and transfected in culture with the desired genetically engineered gene. These cells form a callus from which a new plant can emerge.

Watch the animation about the secretory pathway, and then answer the questions. The drug vinblastine disrupts microtubule polymerization. How would adding vinblastine to a cell affect the constitutive secretory pathway?

Transport vesicles will not be brought to either the Golgi apparatus or the plasma membrane. FEEDBACK: Microtubules are used as tracks for the transport of organelles and vesicles around the cell. Transport vesicles that bud off the endoplasmic reticulum are transported along microtubules to the Golgi apparatus. From the Golgi, vesicles are transported along microtubules to the plasma membrane, where the vesicle contents are released out of the cell.

pinocytosis

Type of endocytosis in which soluble materials are taken up from the environment and incorporated into vesicles for digestion. (Literally, "cell drinking.") (See also endocytosis.) (page 524)

Watch the animation about clathrin, and then answer the questions. Scientists have modified a clathrin molecule so that it still assembles but forms an open-ended lattice instead of a closed spherical cage. How would this clathrin molecule affect endocytosis in cells?

Vesicles cannot form properly without a clathrin cage, thus inhibiting endocytosis. FEEDBACK: Clathrin proteins are responsible for forming the closed spherical cage around the forming vesicle. This cage helps shape and build the vesicle. When clathrin is mutated, it will not form the proper cage and instead will make a flat sheet as an open-ended lattice. This flat sheet will not form the spherical vesicle and so endocytosis will not occur. Any processes that require clathrin to form the vesicle in the cell will be blocked. Other transport mechanisms that do not rely upon clathrin will still function normally.

A single-pass transmembrane protein destined for one of the organelles in the endomembrane system would be marked by what type of signal sequence?

a cleaved N-terminal ER signal sequence and an internal stop-transfer sequence FEEDBACK: Proteins that are destined for the ER lumen have an N-terminal signal sequence—a hydrophobic region that, once cleaved, releases the protein to the interior of the ER. If the protein also contains an internal stop-transfer sequence, the transfer of the protein is halted once it reaches this hydrophobic region, and it remains embedded in the ER membrane. The N-terminal signal sequence is still cleaved to release the N-terminal end from the membrane.

Phagocytosis is a process by which cells do which of the following?

consume large particles, such as microbes and cell debris FEEDBACK: The most dramatic form of endocytosis, phagocytosis, was first observed more than a hundred years ago. In protozoa, phagocytosis is a form of feeding: these unicellular eukaryotes ingest large particles such as bacteria by taking them up into phagosomes. The phagosomes then fuse with lysosomes, where the food particles are digested. Few cells in multicellular organisms are able to ingest large particles efficiently. In the animal gut, for example, large particles of food have to be broken down to individual molecules by extracellular enzymes before they can be taken up by pinocytosis by the absorptive cells lining the gut. Nevertheless, phagocytosis is important in most animals for purposes other than nutrition. Phagocytic cells—including macrophages, which are widely distributed in tissues, and other white blood cells, such as neutrophils—defend us against infection by ingesting invading microorganisms.

What would the final destination be for a protein bearing both an ER sorting signal and a nuclear localization signal?

endoplasmic reticulum FEEDBACK: An endoplasmic reticulum sorting signal directs a protein to the ER while it is still being synthesized, thus an ER sorting signal would supersede a nuclear localization signal. A nuclear localization signal imports a completely synthesized protein from the cytosol to the nucleus.

Watch the animation about the secretory pathway, and then answer the questions. During a pulse-chase experiment with secreted proteins, the proteins are synthesized for a short "pulse" time with radioactive or fluorescent amino acids to label the proteins. During the "chase" period, unlabeled amino acids are added, so any additional proteins synthesized are not labeled. The labeled proteins can then be monitored over time. You complete a pulse-chase experiment to monitor the secretion of a protein from the cell. Which of the following correctly lists the order of locations of the protein during the chase period?

endoplasmic reticulum → transport vesicle → Golgi → transport vesicle → secreted FEEDBACK: Secreted proteins are synthesized at the rough endoplasmic reticulum, where ribosomes can be found. During constitutive secretion, proteins accumulate at random locations in the endoplasmic reticulum membrane network and are packaged into transport vesicles. The vesicles fuse into transport intermediates that travel along microtubule tracks to the Golgi apparatus. From the Golgi, proteins are again packaged into new transport vesicles for travel along additional microtubules to the plasma membrane, where they are secreted to the outside of the cell.

Which cellular compartment acts as the main sorting station for extracellular cargo molecules taken up by endocytosis?

endosomes FEEDBACK: Just as the Golgi network acts as the main sorting station in the outward secretory pathway, the endosomal compartment serves this function in the inward endocytic pathway. The acidic environment of the endosome (pH 5 to 6) plays a crucial part in the sorting process by causing many (but not all) receptors to release their bound cargo. The routes taken by receptors once they have entered an endosome differ according to the type of receptor: (1) most are returned to the same plasma membrane domain from which they came, as is the case for the LDL receptor discussed earlier; (2) some travel to lysosomes, where they are degraded; and (3) some proceed to a different domain of the plasma membrane, thereby transferring their bound cargo molecules across the cell from one extracellular space to another, a process called transcytosis.

Which of the following is a covalent modification that occurs mainly in the ER?

formation of disulfide bonds FEEDBACK: Two major covalent modifications occur in the ER. Certain proteins need disulfide bonds to form between cysteines to stabilize their structure, and some proteins have a branched oligosaccharide added to them, which is then matured in the Golgi into the final structure glycoprotein structure.

Botulism is a potentially fatal foodborne disease caused by the bacterium Clostridium botulinum. C. botulinum produces different toxins, several of which are proteases that cleave neuronal SNARE proteins. What normal process is blocked by cleavage and inhibition of SNARE proteins?

fusion of vesicles with target membranes FEEDBACK: Many different proteins help vesicles dock and fuse with the correct target membrane (see below). Rabs interact with tethering proteins during the docking phase, bringing v-SNAREs and t-SNAREs into close proximity. The SNAREs then intertwine, aiding vesicle fusion with the target membrane. In neurons, the t-SNARE synaptosomal nerve-associated protein 25 (SNAP-25) is important for the fusion of neurotransmitter-containing vesicles with the plasma membrane. Cleavage of SNAP-25 by botulinum toxin prevents vesicle fusion (PeerJ. 2015; 3: e1065) and neurotransmitter release, leading to paralysis. Proteins involved in vesicle docking and fusing with target membranes.

Trypanosomes are single-celled parasites that cause sleeping sickness when they infect humans. Trypanosomes taken from infected humans are known to store the enzymes needed to carry out some of the reactions of glycolysis in an organelle that resembles the peroxisome. In contrast, trypanosomes taken from tsetse flies—the intermediate host—carry out glycolysis entirely in the cytosol.Investigators at a pharmaceutical company decide to follow up on this observation to design a potential new therapeutic. They determine that in trypanosomes from tsetse flies, one of the glycolytic enzymes, phosphoglycerate kinase (PGK), is present entirely in the cytosol, whereas in parasites taken from humans, 90% of the PGK activity is in a peroxisome-like compartment and only 10% is in the cytosol.When the investigators clone the PGK genes, they discover that the parasites have three forms, each of which differs slightly from the others. They design probes that hybridize specifically to the mRNAs from each gene and then use these probes to determine which genes are expressed by trypanosomes from humans (H) and which are expressed by trypanosomes from tsetse flies (F).Shown here is a gel in which mRNAs purified from the two different trypanosomes have been separated by size and exposed to probes that recognize the three different forms of the PGK gene (genes 1, 2, and 3). Based on these results, which gene most likely encodes the peroxisomal form of PGK?

gene 3 FEEDBACK: The peroxisomal form of PGK is found only in the trypanosomes taken from humans. Therefore, the easiest way to determine which band likely represents the peroxisomal form of PGK is to focus on the gene that is expressed only in trypanosomes from humans. Gene 2 is found only in the parasites from flies. Because this form is cytosolic, gene 2 is not the likely culprit. Gene 1 is expressed at low levels in parasites from both humans and flies. Because this gene is present in both samples, it is unlikely responsible for the large differences seen in the localization of PGK. Gene 3 is expressed exclusively in trypanosomes from humans, so it most likely encodes the form of PGK that is localized to the peroxisome-like organelle. It is also expressed at a much higher level than gene 1, which agrees well with the observation that 90% of the PGK activity is present in the peroxisome-like organelle. Because gene 1 is expressed at low levels in the parasite from humans, it is likely responsible for the 10% of PGK activity that is seen in the cytosol.

Lysosomes contain ____________ enzymes that can break down diverse macromolecules, cell parts, and microorganisms.

hydrolytic FEEDBACK: Lysosomes are a compartment in the cell where ingested cell particles, organelles, or macromolecules can be digested for recycling. The macromolecules from ingestion or from these cell parts are broken down into their building blocks by hydrolytic enzymes and are then exported out of the lysosome for reuse.

Insulin is synthesized in the form of a precursor protein that requires cleavage of two different peptide segments before the mature protein is secreted from β cells in the pancreas. The first peptide is removed when the protein enters the lumen of the ER. To find out when the second cleavage event takes place, investigators prepare a pair of antibodies: one recognizes the pro-insulin precursor, the other the mature insulin protein. They tag the antibody that binds to the precursor protein with a red fluorescent marker; the antibody that binds to mature insulin is tagged with a green fluorescent marker. When both markers are present, the sample fluoresces yellow. The investigators then incubate an isolated β cell with both antibodies at the same time and monitor the fluorescence in its various membrane-bound compartments. The data are shown in the table below. Based on these observations, where is the second peptide removed from the pro-insulin precursor protein?

immature secretory vesicles FEEDBACK: Correct. The appearance of yellow fluorescence in the immature secretory vesicles indicates that both the precursor protein and mature insulin are present, which suggests that the cleavage is taking place here.

Proteins encoded by nuclear genes and destined for the mitochondrial matrix are

in possession of a signal sequence for targeting to the mitochondria. FEEDBACK: Mitochondrial proteins that are encoded by genes in the nucleus are synthesized on free ribosomes in the cytosol. A signal sequence in the proteins is recognized by import machinery that transports the unfolded protein across both membranes at once.

transformation

process by which cells take up DNA molecules from their surroundings and then express the genes present on that DNA (page 338)

Nuclear pores restrict larger molecules from traversing the membrane due to their

interwoven meshwork of protein fibrils. FEEDBACK: A nuclear pore is a large passage in the nuclear membrane whose opening is filled with protein fibrils that allow passage of small, water-soluble molecules. Larger molecules are impeded from entering by this meshwork of protein fibrils (unless they have a nuclear localization or export sequence).

Watch the animation on receptor-mediated endocytosis, and then answer the questions. Which of the following components of receptor-mediated endocytosis of LDL is incorrectly matched with its function?

lysosome: releases LDL from the receptor FEEDBACK: When LDL binds to the specific LDL receptor on the plasma membrane, an adaptin binds to the receptor on the cytosol side. Adaptin recruits clathrin proteins, which form the coated vesicle. After vesicle formation, the clathrin is released and the naked transport vesicle is transported to the endosome. The endosome is the specific compartment where the LDL is released from the receptor as a result of the compartment's low pH. The empty LDL receptors are then recycled back to the plasma membrane, where the process can repeat. The LDL particles travel from the endosome to the lysosome where the LDL is broken down, finally releasing cholesterol to the cytosol for use in the cell.

Fully folded proteins can be transported into which of the following organelles?

nucleus FEEDBACK: Transporters in the membranes of mitochondria and chloroplasts unfold the protein during transfer. Proteins destined for the endoplasmic reticulum are transported as they are being synthesized, thus the proteins are unfolded. Nuclear transport occurs on folded proteins.

Which of the following compartments receives proteins directly from cytosol?

peroxisomes FEEDBACK: Proteins destined for the nucleus, mitochondrion, peroxisomes, and chloroplasts are transported from the cytosol. Proteins destined for the

Which of the endocytic pathways involves the ingestion of large particles or microorganisms and is performed mainly by specialized cells?

phagocytosis FEEDBACK: The endocytic pathways are phagocytosis and pinocytosis. Pinocytosis involves the intake of molecules and fluid and is active in all cells. Phagocytosis is a process by which microorganisms or large particles are ingested by a specialized phagocytic cell, such as those in the immune system.

Which of the following pathways helps selectively concentrate substances to be ingested by their binding to proteins on the cell surface?

receptor-mediated endocytosis FEEDBACK: Receptor-mediated endocytosis is a form of pinocytosis where specific molecules are concentrated on the cell surface by their binding to receptors. The receptors are then taken up in a clathrin-coated vesicle for delivery to the endosome.

When making a DNA library from a starting material of mRNA, which enzyme is first required to copy the mRNA molecules into DNA molecules?

reverse transcriptase FEEDBACK: Correct. This same type of enzyme allows RNA viruses such as HIV to replicate their genomes and integrate into a host cell's DNA

In a typical human secretory cell, which of the following membranes has the largest surface area?

rough ER FEEDBACK: On average, the membrane-enclosed organelles together occupy nearly half the volume of a eukaryotic cell, and in a typical mammalian cell, the area of the endoplasmic reticulum membrane is 20 to 30 times greater than that of the plasma membrane. This organelle is folded over to form an extensive maze of interconnected spaces.Cells can adjust the size of their ER to accommodate the volume of proteins entering the secretory pathway. So, in cells specialized for secretion, the ER can expand and, on its own, compose about half of the total membrane present in the cell.

In eukaryotic cells, phospholipids are synthesized by enzymes bound to which of the following?

the cytosolic face of the endoplasmic reticulum FEEDBACK: Correct. New phospholipids are added to the ER membrane asymmetrically. Some of the newly made phospholipids are subsequently moved from the cytosolic monolayer to the other half of the bilayer so that the membrane can grow evenly.

On what side of the plasma membrane are the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids located?

the extracellular side FEEDBACK: Correct. The sugars on plasma membrane glycolipids, glycoproteins, and proteoglycans all face the cell exterior, where they form a carbohydrate layer or glycocalyx that coats the surface of the cell.

Multipass transmembrane proteins can form pores across the lipid bilayer. The structure of one such channel is shown in the diagram. In this figure, what do the areas shown in red represent?

the hydrophilic side chains of the transmembrane α helices FEEDBACK: Small water-soluble molecules can pass through the water-filled pore formed by the hydrophilic side chains of the transmembrane helices, shown in red.

Which membrane-enclosed organelles most likely evolved in a similar manner?

the nucleus and the ER FEEDBACK: Nuclear membranes and the ER likely arose through invagination of the plasma membrane. In modern bacteria and archaea, a single DNA molecule is typically attached to the plasma membrane. It is possible that, in a very ancient anaerobic archaeon, the plasma membrane, with its attached DNA, could have invaginated and, in subsequent generations, formed a two-layered envelope of membrane completely surrounding the DNA. This envelope is presumed to have eventually pinched off completely from the plasma membrane, ultimately producing a nuclear compartment. Other portions of the invaginated membrane may have formed the ER, which would explain why the space between the inner and outer nuclear membranes is continuous with the ER lumen.Mitochondria and chloroplasts are thought to have evolved from bacteria that were engulfed by primitive eukaryotic cells with which they initially lived in symbiosis. These cells already had a nucleus when they acquired these symbiotic inhabitants.

In the α helices of transmembrane proteins, the hydrophobic side chains face which direction?

the outside of the membrane-spanning helix FEEDBACK: Correct. This arrangement allows the exposed hydrophobic side chains of the α helix to interact with the hydrophobic tails of the lipid bilayer.

In a classic experiment designed to study nuclear transport, investigators added a dye molecule to the subunits of a protein called nucleoplasmin, which is involved in chromatin assembly. They then injected the intact protein or combinations of its subunits into the cytosol of a frog oocyte or into its nucleus.The results of the experiment are shown in the diagram, where red indicates the location of the labeled protein. Based on these results, which part of the nucleoplasmin protein bears a nuclear localization signal?

the tail only FEEDBACK: Before nuclear pore complexes were well understood, it was unclear whether nuclear proteins diffused passively into the nucleus and accumulated there by binding to nuclear components, such as the chromosomes, or they were actively imported and accumulated regardless of their affinity for nuclear components. To address this question experimentally, nuclear proteins were labeled and injected into the nucleus and cytosol and their localization recorded.In this experiment, investigators tracked the localization of nucleoplasmin, a protein complex that consists of five identical subunits, each with a distinct head and tail portion.Focusing on the results of the experiments in which the protein preparations were injected into the cytosol (right-hand column), it can be seen that only the proteins that include at least one tail get transported into the nucleus. The "heads only" preparation remains in the cytoplasm.These results suggest that the nuclear localization signal is present in the nucleoplasmin tail. They also confirm that proteins must be actively and selectively transported into the nucleus and do not enter via passive diffusion. If proteins could diffuse passively into the nucleus, then the nucleoplasmin heads should have also made it into the nucleus. In fact, all of the labeled proteins injected into the nucleus would have been able to diffuse into the cytosol.

Through which of the following do proteins travel from one cisterna to the next in the Golgi apparatus?

transport vesicles that bud from one cisterna and fuse with the next FEEDBACK: Each Golgi stack has two distinct faces: an entry, or cis, face, which is adjacent to the ER, and an exit, or trans, face, which points toward the plasma membrane. The outermost cisterna at each face is connected to a network of interconnected membranous tubes and vesicles. Soluble proteins and pieces of membrane enter the cis Golgi network via transport vesicles derived from the ER. The proteins travel through the cisternae in sequence in two ways: (1) by means of transport vesicles that bud from one cisterna and fuse with the next, and (2) by a maturation process in which the Golgi cisternae themselves migrate through the Golgi stack. Proteins finally exit from the trans Golgi network in transport vesicles destined for either the cell surface or another organelle of the endomembrane system.

How are newly made lipids supplied to the plasma membrane?

via the constitutive pathway of exocytosis FEEDBACK: In all eukaryotic cells, a steady stream of vesicles buds from the trans Golgi network and fuses with the plasma membrane in the process of exocytosis. This constitutive exocytosis pathway supplies the plasma membrane with newly made lipids and proteins, enabling the plasma membrane to expand prior to cell division and refreshing old lipids and proteins in nonproliferating cells.The regulated exocytosis pathway also adds phospholipids to the plasma membrane; however, this pathway only operates in cells specialized for secretion

In a lipid bilayer, where do lipids rapidly diffuse?

within the plane of their own monolayer FEEDBACK: Correct. The lipid bilayer is a two-dimensional fluid in which phospholipids rapidly diffuse within the plane of their own monolayer.

The DNA polymerase enzyme used in PCR is different from the one used in human cells because it is able to

withstand the high temperatures needed for denaturation. FEEDBACK: PCR uses a polymerase that was isolated from a thermophilic bacterium. This enzyme can withstand the high heat needed to denature the DNA strands in step one of PCR.

Watch the animation about leukocyte migration, and then answer the questions. Carbohydrates on the surface of leukocytes play an important role in responding to infection or inflammation. Place the following steps of the response in the correct order.

1. Cytokines are released at sites of infection or inflammation and stimulate endothelial 2. Endothelial cells express selectins on their plasma membrane 3. Selectins bind to carbohydrates on the surface of leukocytes, causing them to stick 4. Leukocytes roll along vessel walls 5. Leukocytes crawl out of vessel into adjacent tissue FEEDBACK: At sites of infection, cytokines are released that stimulate local endothelial cells of blood vessels to express selectin proteins on their plasma membrane. Selectin proteins bind specific carbohydrates on the surface of cells, including leukocytes (white blood cells). When selectin binds to carbohydrate on a leukocyte, the binding is relatively weak, so contacts are made and broken. This leads to a slow rolling of the leukocyte along the blood vessel wall. At certain points, the leukocytes can adhere tightly and squeeze between the endothelial cells to pass out of the vessel and into the affected tissue where they help respond to the infection or inflammation.

Growth hormone, secreted by the pituitary gland, is important for organismal growth. Individuals with two mutated, nonfunctional copies of the growth hormone gene exhibit dwarfism. Early treatments relied on injecting patients with growth hormone isolated from the pituitary glands of cadavers, an expensive procedure that unfortunately infected some recipients with prion particles that caused Creutzfeldt-Jakob disease. Researchers sought a safer source of growth hormone via the production of human growth hormone in E. coli bacteria. Put in order the series of steps undertaken to create human growth hormone producing transgenic E. coli.

1. Isolate mRNA from pituitary gland. 2. Convert mRNA into cDNA 3. Insert cDNAs into plasmids, creating cDNA library. 4. Transform plasmids into E. coli cells. 5. Detect E. coli containing growth hormone DNA via hybridization FEEDBACK: Correct. This is the general approach to cloning a cDNA for a particular gene via plasmid cloning.

The restriction enzyme HindIII cuts DNA at the sequence AAGCTT. In the human genome, which is 3.2 × 109 base pairs long, approximately how often will this target sequence be encountered?

780,000 times FEEDBACK: Correct. As the restriction enzyme HindIII cuts DNA at the sequence AAGCTT, this enzyme would cut human DNA approximately 780,000 times.The HindIII target sequence would be expected to occur at random about once every 4000 nucleotide pairs (46 = 4096). The human genome has 3.2 × 109 nucleotide pairs, so a HindIII target sequence should occur on average about 780,000 times (3 × 109 ÷ 4096 = 781,250).

Approximately how many double-stranded DNA molecules will be formed from one starting molecule after three rounds of PCR?

8 FEEDBACK: After the first cycle of a PCR reaction, each starting target DNA molecule serves as the template for generating two new daughter molecules, comparable to DNA replication in cell. The second cycle will then duplicate each of the molecules from the first cycle, producing four total molecules, and the third cycle duplicates each of those four molecules into eight.

You have the cDNA for a human gene that encodes a protein that can be used therapeutically. You want to produce this protein in yeast cells to provide a safe, reliable supply for patients. You have a yeast expression vector; this is a plasmid that contains a promoter followed by a multi-cloning site (MCS) where genes can be inserted. The MCS contains unique sites for both EcoRI and HindIII restriction enzymes. Your cDNA is flanked by EcoRI sites, so you cut the plasmid with EcoRI and ligate in your cDNA. Unfortunately, the cDNA can ligate in either direction, producing two different final constructs. Since you want the gene to be transcribed, the orientation relative to the promoter is important. To determine the orientation of the insert, you cut the plasmid with HindIII and determine fragment sizes. The 3000-base-pair (3 kb) vector contains a unique EcoRI site and a HindIII site 20 base pairs away. Your 1000-base-pair-long cDNA also contains a HindIII site 200 base pairs from the start. In the correct orientation, the initiating methionine codon (ATG) is closer to the promoter relative to the incorrect orientation. After cutting the plasmid with HindIII, what sizes (in base pairs) are expected for plasmids containing a cDNA in the correct orientation for protein expression? A schematic of the expression vector and insert is shown here. Note that sizes are not drawn to scale, but relative orientations are correct.

820, 3180 FEEDBACK: Correct. These are the expected fragment sizes. Note that they add up to 4000 base pairs.

Investigators are attempting to clone a gene that is bracketed by the following 5'-to-3' sequence: ATGAAATCTACGTTTCAC......CCCCCAGTACCCCCCTTA Which of the following pairs of primers could be used to direct the amplification of this gene? (All sequences are written 5' to 3'.)

ATGAAATCTACGTTTCAC and TAAGGGGGGTACTGGGGG FEEDBACK: Correct. The first primer would hybridize with the 3' end of the strand that is complementary to the sequence shown. This "forward" primer (with a sequence ATGAAATCTACGTTTCAC) would be extended to the right (in the 5'-to-3' direction). The second primer would hybridize to the 3' end of the sequence shown. This "reverse" primer, which would have the sequence TAAGGGGGGTACTGGGGG, would be extended to the left (also in the 5'-to-3' direction).

Shown is a schematic diagram of a membrane phospholipid. Which segment will always carry a negative charge?

B FEEDBACK: (B) represents the phosphate group, which is always negatively charged.

Watch the animation about the FRAP technique, and then answer the questions. The lamin B receptor is found in the inner membrane of the nuclear envelope. It connects the nuclear envelope to the heterochromatin (chromosome) and nuclear lamina proteins, which provide structure to the nucleus. In normal cells, the lamin B receptor protein is stably locked in place by these interactions and shows very little movement. Infection of cells by the Herpes Simplex Virus Type I (HSV-1) can disrupt the lamin B receptor interactions when the virus capsids exit from the nucleus by budding through the inner nuclear membrane of the nucleus. This causes some of the lamin B receptor to move between the inner nuclear membrane and the ER membrane.FRAP was completed for lamin B or a control protein under different conditions. Match the three cellular treatments with the correct FRAP graph.

B, C, A FEEDBACK: The lamin B receptor protein is anchored to the inner nuclear envelope by interactions with the heterochromatin and the nuclear lamina proteins. Under normal conditions, the protein will move very little and there will be little fluorescence recovery over time, as seen in graph A. The Herpes Simplex Virus Type I will form capsids in the nuclei of infected cells. As these capsids bud through the inner nuclear envelope, inner nuclear envelope proteins like lamin B receptor are moved out from the nuclear membrane to the ER. These proteins are then recycled back to the inner nuclear envelope. This leads to some movement and subsequently some recovery of fluorescence over time, as seen in graph C. The control cells with a protein known to cycle rapidly will show a rapid recovery in fluorescence, as seen in graph B.

Watch the animation about leukocyte migration, and then answer the questions. Mutation in the hemoglobin gene can cause sickle-cell anemia. The defective protein found in sickle-cell anemia causes red blood cells to "sickle"—become a misshapen C shape. These misshapen cells abnormally stick to each other and can become trapped by leukocytes (white blood cells) that are rolling or paused on the endothelial cells lining the vessel. This causes blockages of small blood vessels, causing severe pain and strokes called vaso-occlusive crisis. A new drug that binds and blocks selectin proteins is in phase III clinical trials to test for improvement in patients' symptoms. Why might this be an effective treatment for vaso-occlusive crisis?

Blocking selectins would block the ability of selectin to bind leukocytes, so leukocytes would be less likely to move slowly along the vessel wall and cause a blockage of red blood cells. FEEDBACK: Selectins are expressed by the endothelial cells lining veins. The selectins bind to carbohydrates on the surface of leukocytes (white blood cells) to slow the movement of the leukocytes through the vein. The leukocytes roll along the vessel wall before squeezing between endothelial cells into the surrounding tissue. A drug that can bind and block selectin proteins would lessen the number of leukocytes bound to the vessel wall. There would then be fewer leukocytes to trap the deformed red blood cells and the red blood cells should continue to move through the blood vessels. Fewer blockages would lead to less pain and a reduced risk of strokes that occur in vaso-occlusive crisis.

To study the structure of a particular membrane protein, the target protein is usually removed from the membrane and separated from other membrane proteins. Shown below are three different proteins associated with the cell membrane. Treatment with high salt would release which protein or proteins from the bilayer?

C FEEDBACK: Correct. Treatment with high salt or changing the pH of the solution can disrupt protein-protein interactions. High salt would therefore release this peripheral membrane protein from the bilayer.The other proteins are integral membrane proteins; their attachment to the membrane would not be altered by salt alone.

The diffusion of an integral membrane protein is studied by fluorescence recovery after photobleaching (FRAP). In this procedure, the protein of interest is labeled with a fluorescent marker, and the fluorescence in a small patch of membrane is then irreversibly "bleached" by a pulse of light from a focused laser. The time it takes for fluorescence to return to the bleached membrane patch provides a measure of how rapidly unbleached, fluorescently labeled proteins diffuse through the bilayer into the area. This "recovery" is plotted on a curve that shows fluorescence over time.For one protein, which acts as a receptor for an extracellular signal molecule, stimulation by its signal ligand causes the receptor to interact with other membrane proteins, forming a large protein signaling complex.Shown here is the FRAP result for the unstimulated receptor. Which of the following curves would most likely represent the behavior of the receptor once it has been activated by its signal molecule?

C FEEDBACK: Correct. With the receptor protein tied up in a large signaling complex, its diffusion through the membrane is slowed to the extent that the fluorescence does not return to its initial levels in that patch of membrane within the 100-second interval shown.

More than 500 human genetic diseases are caused by haploinsufficiency, a heterozygotic condition in which the wild-type allele cannot produce enough normal protein to make up for the loss of functional protein expression from the mutated allele to get a normal phenotype. What might be an approach for treating haploinsufficiency disorders in non-human disease models?

CRISPR targeting wild-type gene with inactive Cas9 fused to a transcription activator FEEDBACK: Correct. This would boost production of the wild-type gene, leading to more protein production as shown in the image below. This approach was recently used to treat a genetic cause of obesity in a mouse model (Matharu et al. 2019, Science vol. 363). Catalytically inactive Cas9 fused to a transcriptional activator can increase gene expression from a gene specified by the guide RNA (in blue).

Proteins destined for the Golgi apparatus, endosomes, lysosomes, and even the cell surface must pass through which organelle?

ER FEEDBACK: Unlike the nucleus, mitochondria, and peroxisomes, the ER serves as an entry point for proteins destined for other organelles, as well as for the ER itself. Proteins destined for the Golgi apparatus, endosomes, and lysosomes, as well as proteins destined for the cell surface, all first enter this extensive system of membranes from the cytosol. Once inside the ER lumen, or embedded in the ER membrane, individual proteins will not re-enter the cytosol during their onward journey. They will instead be ferried by transport vesicles from organelle to organelle within the endomembrane system, or to the plasma membrane.

Shown here is a drawing of a cell from the lining of the mammalian intestine. Label its organelles by dragging the labels on the right to the targets in the image.

FEEDBACK: In a eukaryotic cell, the major membrane-enclosed organelles are surrounded by the cytosol, which is enclosed by the plasma membrane. The nucleus, which is surrounded by a double membrane perforated by nuclear pores, is generally the most prominent organelle in the cell. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER), a system of interconnected membranes that often extends throughout most of the cell. Large areas of the ER have ribosomes attached to its cytosolic surface via the proteins they are synthesizing, which are inserted into the ER membrane. Ribosomes that are synthesizing cytosolic proteins remain unattached from the ER, floating free in the cytosol. The Golgi apparatus, which looks like a flattened stack of membranous discs, is usually situated near the nucleus (but is not continuous with the nuclear envelope, as is the ER). Small organelles called endosomes, often located near the plasma membrane, sort ingested material, some of which is passed on to spherical lysosomes—either by fusion with preexisting lysosomes or by a maturation process that converts the endosome into a classical lysosome. Peroxisomes contain enzymes that produce hydrogen peroxide. Mitochondria are surrounded by a double membrane, with an inner membrane that is highly folded.

Which organelle receives proteins and lipids from the endoplasmic reticulum, modifies them, and then dispatches them to other destinations in the cell?

Golgi apparatus FEEDBACK: The Golgi apparatus, which is usually situated near the nucleus, receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cell. Transport from the ER to the Golgi apparatus—and from the Golgi apparatus to other compartments of the endomembrane system—is carried out by the continual budding and fusion of transport vesicles. Proteins entering the Golgi can either move onward through the Golgi stack or, if they contain an ER retention signal, be returned to the ER; proteins exiting from the Golgi are sorted according to whether they are destined for lysosomes (via endosomes) or for the cell surface.

Watch the animation about leukocyte migration, and then answer the questions. The following graphs show the number of adherent leukocytes found on the blood vessel wall in control conditions and after adding a selectin inhibitor, which blocks the function of selectin. Which of the following graphs correctly shows the effect of a selectin inhibitor on adherence of leukocytes to the vessel wall?

Graph B FEEDBACK: A drug that inhibits selectin function on the endothelial cells will inhibit the ability of the selectin to bind to carbohydrates on the surface of leukocytes. Graph B shows fewer leukocytes adhering to endothelial cells because the selectin is blocked. The leukocytes still express the carbohydrates on their surface, but the selectin on endothelial cells is blocked from binding. This leads to fewer leukocytes being bound and can lead to fewer red blood cells trapped in sickle-cell anemia. The drug GMI-1070 is currently in phase III clinical trials to determine if it can help treat vaso-occlusive crisis. Graph B in Part 3 comes from an earlier study of GMI-1070 that showed that the drug could effectively block leukocyte binding to endothelial cells, leading to symptom improvement in mice (Chang, J. et al., Blood 116:1779-1786, 2010).

When scientists were first studying the fluidity of membranes, they did an experiment using hybrid cells. Certain membrane proteins in a human cell and a mouse cell were labeled using antibodies coupled with differently colored fluorescent tags. The two cells were then coaxed into fusing, resulting in the formation of a single, double-sized hybrid cell. Using fluorescence microscopy, the scientists then tracked the distribution of the labeled proteins in the hybrid cell. Which best describes the results they saw and what they ultimately concluded?

Initially, the mouse and human proteins were confined to their own halves of the newly formed hybrid cell, but over time, the two sets of proteins became evenly intermixed over the entire cell surface. This suggests that proteins, like lipids, can move freely within the plane of the bilayer. FEEDBACK: Because a membrane is a two-dimensional fluid, many of its proteins, like its lipids, can move freely within the plane of the bilayer. This lateral diffusion was initially demonstrated by experimentally fusing a mouse cell with a human cell to form a large, hybrid cell and then monitoring the distribution of certain mouse and human plasma membrane proteins. At first, the mouse and human proteins are confined to their own halves of the newly formed hybrid cell, but within half an hour or so, the two sets of proteins become evenly mixed over the entire cell surface, as shown below. Based on observations of LD. Frye and M. Eddin, J. Cell Sci. 7:319-335, 1970. To monitor the movement of the selected proteins, the cells were labeled with antibodies that bind to either human or mouse proteins; these antibodies are coupled to two different fluorescent tags—shown here in red and blue—so that the proteins to which the antibodies bind can be distinguished in a fluorescence microscope.

If a protein with a specific activity is isolated, how can the encoding gene be identified?

It can be identified by mass spectrometry followed by gene database searching. FEEDBACK: Proteins of interest can be identified using mass spectrometry to determine many short amino acid sequences read from the protein. These sequences can then be compared to a database of all known protein-coding genes to identify the gene that encodes this protein.

As a polypeptide is being translocated across the membrane of the endoplasmic reticulum, a stop-transfer sequence can halt the process. What eventually becomes of this stop-transfer sequence?

It forms an α-helical membrane-spanning segment of the protein. FEEDBACK: For some proteins, transfer into the ER is halted by a sequence of hydrophobic amino acids, a stop-transfer sequence, within the polypeptide chain. When this sequence enters the protein translocator, the growing polypeptide chain is released sideways into the lipid bilayer. The N-terminal ER signal sequence is eventually cleaved off, but the stop-transfer sequence remains in the bilayer, where it forms an α-helical membrane-spanning segment whose hydrophobic side chains interact with the hydrophobic lipid tails within the bilayer, thereby anchoring the protein in the membrane.Meanwhile, protein synthesis on the cytosolic side continues to completion. As a result, the protein ends up as a single-pass transmembrane protein inserted in the membrane with a defined orientation—the N-terminus on the lumenal side of the lipid bilayer and the C-terminus on the cytosolic side.

What is true of a reporter gene?

It is a gene whose product can be visually monitored. & It can reveal when a gene is expressed. FEEDBACK: Correct. A reporter gene can reveal when and where a gene is expressed because the gene product can be visually monitored. Green fluorescent protein (GFP), a luminescent protein from jellyfish, is one popular reporter gene. Recombinant DNA techniques can be used to fuse GFP to the promoter or other regulatory sequences of other genes, allowing researchers to easily view expression patterns.

How does the cortex of a typical animal cell differ from that of a mature red blood cell?

It is richer in actin and in the motor protein myosin. It allows the cell to move. It allows the cell to selectively take up material from the environment. FEEDBACK: Correct. Whereas red blood cells need their cortex mainly to provide mechanical strength as they are pumped through blood vessels, other animal cells also use their cortex to selectively take up materials from their environment, to change their shape, and to move.

How does the inclusion of cholesterol affect animal cell membranes?

It tends to make the lipid bilayer less fluid. FEEDBACK: Correct. This stiffening makes the bilayer less flexible as well as less permeable.

How does automated Sanger sequencing differ from the original method?

It uses a mixture of chain-terminating nucleotides, each with its own label. FEEDBACK: Correct. Automated Sanger sequencing differs from the original method in that it uses a mixture of chain-terminating nucleotides, each with its own label. These generate a mixture of DNA products of different lengths, each containing a chain-terminating nucleotide of a different color. These products are separated by electrophoresis and the color of the fluorescent labels is read by a computer.

In 1976, a group of scientists reported the characterization of a heat-stable DNA polymerase from the hyperthermophile bacterium Thermus aquaticus (Chien A, Edgar DB, Trela JM (1976). J. Bacteriol. 127 (3): 1550-7). This enzyme had a temperature optimum of 80°C and was stable for several minutes at even higher temperatures. Unbeknownst to the researchers at the time, about a decade later this heat-stable DNA polymerase would find an important application in which technique?

PCR FEEDBACK: Correct. Polymerase chain reaction, PCR, is an in vitro cloning technique. Template DNA undergoes rounds of (1) denaturation at 95°C to disrupt H bonding between complementary bases of the double-stranded DNA molecule, (2) primer annealing to single-stranded DNA, and (3) DNA synthesis using heat-stable Thermus aquaticus DNA polymerase, as summarized in the figure below.

Which statements are true about the differences between phospholipids and detergents?

Phospholipids form bilayers in water, whereas detergents tend to form micelles., Detergents are shaped like cones, whereas phospholipids are more cylindrical., Phospholipids have two hydrocarbon tails, whereas detergents have just one. FEEDBACK: Correct. Detergents differ from membrane phospholipids in that they have only a single hydrophobic tail. Because they have one tail, detergent molecules are shaped like cones; in water, this shape drives these amphipathic molecules to form small clusters called micelles, rather than forming a bilayer as do the phospholipids, which—with their two tails—are more cylindrical.

Which proteins play a central role in the fusion of a vesicle with a target membrane?

SNAREs FEEDBACK: Correct. SNARE proteins on the vesicle interact with SNARE proteins in the target membrane to help vesicles dock. By winding around each other tightly, SNARE proteins pull the membrane bilayers close enough to allow their lipids to flow together.

How does a bacterium protect its DNA from cleavage by its own restriction nucleases?

The bacterium chemically modifies its own DNA sequences, thereby preventing recognition by the nucleases. FEEDBACK: Correct. By chemically modifying their own DNA sequences, bacteria prevent recognition of their own DNA by their own nucleases. Bacteria methylate their DNA to prevent it from being recognized by restriction nucleases.

In 1925, scientists exploring how lipids are arranged within cell membranes performed a key experiment using red blood cells. Using benzene, they extracted the lipids from a purified sample of red blood cells. Because these cells have no nucleus and no internal membranes, any lipids they obtained were guaranteed to come from the plasma membrane alone. The extracted lipids were floated on the surface of a trough filled with water, where they formed a thin film. Using a movable barrier, the researchers then pushed the lipids together until the lipids formed a continuous sheet only one molecule thick. The researchers then made an observation that led them to conclude that the plasma membrane is a lipid bilayer. Which of the following would have allowed the scientists to come to this conclusion?

The extracted lipids covered twice the surface area of the intact red blood cells. FEEDBACK: Correct. The researchers found that the extracted lipids occupied twice the area of the original, intact cells. Additional experiments showed that lipids can spontaneously form bilayers when mixed with water. Together, these observations suggest that in an intact cell membrane, the lipid molecules double up to form a bilayer—an arrangement that has a profound influence on cell biology.

When the transport vesicle shown below fuses with the plasma membrane, which monolayer will face the cell cytosol?

The orange monolayer will face the cytosol. FEEDBACK: Correct. The cytosolic monolayer will always face the cytosol, whether the vesicle is moving between organelles or fusing with the plasma membrane.

Consider the following image of a stained agarose gel representing the separation of a piece of DNA that has been digested by a series of restriction enzymes. Assume no digest fragment is of identical size to another digest fragment. Based on all data presented in the stained agarose gel, which of the following statements is correct?

The original piece of DNA was circular and was cut by EcoRI twice and HindIII only once. FEEDBACK: Correct. The original piece of DNA must have been circular and was cut by EcoRI twice because there are two different-sized fragments of DNA observed on the gel. Similarly, the circular DNA was cut by HindIII only once, which is why there is only one size DNA fragment observed on the gel. The fourth lane provides the conclusive nature of this result, as there are three bands present: EcoRI cut twice and HindIII only once in the middle of one of the two EcoRI-released fragments.

Watch the animation about the FRAP technique, and then answer the questions. The FRAP technique occurs in a series of steps. Select every statement that correctly describes a step in the FRAP procedure.

The relative mobility of the fluorescently labeled molecule is measured. The molecule of interest is fluorescently labeled. FEEDBACK: The FRAP technique measures the relative mobility of a fluorescently labeled molecule of interest. The molecule must be fluorescently labeled so that its location can be measured. The fluorescent marker is then bleached in a small area of the cell, not across the whole cell. The movement of unbleached molecules into the bleached area is measured and quantified. The markers that are bleached are irreversibly bleached and cannot be repaired in the cell.

What happens to proteins with no signal sequence that are made in the cytosol?

They remain in the cytosol. FEEDBACK: The fate of any protein molecule synthesized in the cytosol depends on its amino acid sequence, which can contain a sorting signal that directs the protein to the organelle in which it is required. Proteins that lack such signals remain as permanent residents of the cytosol; those that possess a sorting signal move from the cytosol to the appropriate organelle. Different sorting signals direct proteins into the nucleus, mitochondria, chloroplasts (in plants), peroxisomes, and the ER.Signal sequences are both necessary and sufficient to direct a protein to a particular destination. This has been shown by experiments in which the sequence is either deleted or transferred from one protein to another by genetic engineering techniques. Deleting a signal sequence from an ER protein, for example, converts it into a cytosolic protein, demonstrating that a signal sequence is required to enter the ER. Conversely, placing an ER signal sequence at the beginning of a cytosolic protein redirects the protein to the ER, which shows that a signal sequence is sufficient to direct any protein to the ER.

Why are transgenic animals used to model human diseases in which mutant genes play a major part?

Transgenic approaches in humans are unethical. FEEDBACK: Correct. Transgenic animals are used to model human diseases in which mutant genes play a major part because using transgenic approaches in humans would be unethical. Human genes are as prone to mutation and easy to manipulate as those of other mammals, and our germ-line cells can be genetically altered.

Watch the animation on PCR, and then answer the questions. On your first attempt to run a PCR, you realize you forgot to add one of the two primers. The graduate student you work with suggests you throw away your reaction and start over. Why?

You will get a small amount of product that is not correct. FEEDBACK: Correct. The product will be a long, single-stranded product in very small amounts. Additionally, only one DNA strand will be copied in each round of PCR, so no amplification will occur.

Watch the animation on the CRISPR system, and then answer the questions. Scientists are working to develop the CRISPR-Cas9 system to treat human disease. One of the first clinical trials in humans will use CRISPR to treat β thalassemia. People with β thalassemia have low levels of hemoglobin and cannot get enough oxygen to all their tissues. The treatment is designed to cleave the gene BCL11A that is important for repressing fetal hemoglobin, which is usually only produced in infancy. Cleaving this gene will prevent expression of the inhibitor BCL11A so that fetal hemoglobin can be produced even in adults, alleviating the symptoms. Which of the following are important components added to cells from β thalassemia patients in order to treat the disorder?

a guide RNA that recognizes the BCL11A gene, the Cas9 enzyme FEEDBACK: Correct. To edit the BCL11A gene, a guide RNA that recognizes the gene and the Cas9 enzyme are needed

Which membrane would show a more rapid recovery of fluorescence in a FRAP study?

a membrane containing a larger proportion of unsaturated fatty acids FEEDBACK: Correct. FRAP (fluorescence recovery after photobleaching) is a method used to measure the fluidity of a cell membrane.Membranes containing a larger proportion of unsaturated fatty acids are indeed more fluid; hence, they should exhibit a more rapid recovery in FRAP.

Nucleic acid hybridization, which can be used to detect any given DNA or RNA sequence in a mixture of nucleic acid fragments, relies on what factor?

a single strand of DNA or RNA forming a double helix with another nucleic acid strand of the complementary nucleotide sequence. FEEDBACK: Correct. A single strand of DNA or RNA will form a double helix with another nucleic acid strand of the complementary nucleotide sequence.

Using PCR, after 30 rounds of replication beginning with a single template molecule of double-stranded DNA, how many copies of the target sequence would you expect?

about 1 billion FEEDBACK: Correct. Using PCR, after 30 rounds of replication beginning with a single template molecule of double-stranded DNA, about 1 billion copies of the target sequence would be expected (230 = about 1 billion). Each cycle of PCR doubles the amount of DNA produced in the previous cycle.

Watch the animation on the CRISPR system, and then answer the questions. The CRISPR-Cas9 system was discovered in bacteria, which use it as a defense mechanism against viral infections. Scientists have experimentally modified this system to work in other organisms. Which of the following statements correctly describe an application of the CRISPR system?

activation of transcription of a specific gene, repression of transcription of a specific gene, gene editing of a specific gene FEEDBACK: Correct. CRISPR can be used to edit a gene and activate or repress a specific gene.

Watch the animation about lipids and the lipid bilayer, and then answer the questions. Which of the following would be most likely to disrupt lipid bilayer formation?

addition of a phosphate to the end of the lipid tail FEEDBACK: Correct. Addition of a negatively charged phosphate to the hydrophobic lipid tail would likely disrupt the formation of the lipid bilayer.

Which of these strategies do prokaryotic cells use to isolate and organize their chemical reactions?

aggregating proteins into multicomponent complexes that form biochemical subcompartments with distinct functions FEEDBACK: Correct. The formation of these subcompartments can involve the participation of scaffold proteins that bring together components involved in a particular reaction sequence, such as DNA synthesis.

Which term correctly describes the entire phospholipid molecule?

amphipathic FEEDBACK: Correct. Phospholipids contain both a hydrophilic and hydrophobic component and are therefore amphipathic. This property allows them to form bilayers in water, where the hydrophilic portions interact with the aqueous environment on either side of the membrane, while the hydrophobic portions are shielded from water in the bilayer's interior.

Restriction enzymes cut DNA

at a specific sequence, whether in cells or in a test tube FEEDBACK: Restriction enzymes are enzymes that recognize and cut specific sequences. While matching sequences are protected in the host bacterium's genome, this is an effective way to cleave any DNA of foreign origin. Restriction enzymes are widely used in laboratories to cut DNA at a specific sequence, regardless of DNA's origin.

Animals exploit the phospholipid asymmetry of their plasma membrane to distinguish between live cells and dead ones. When animal cells undergo a form of programmed cell death called apoptosis, phosphatidylserine—a phospholipid that is normally confined to the cytosolic monolayer of the plasma membrane—rapidly translocates to the extracellular, outer monolayer. The presence of phosphatidylserine on the cell surface serves as a signal that helps direct the rapid removal of the dead cell.How might a cell actively engineer this phospholipid redistribution?

by activating a scramblase and inactivating a flippase in the plasma membrane FEEDBACK: Correct. During programmed cell death (apoptosis), the scramblase that transfers random phospholipids from one monolayer of the plasma membrane to the other is fully activated. This causes phosphatidylserine—initially deposited in the cytosolic monolayer—to become distributed to both halves of the bilayer. At the same time, the flippase that would normally transfer phosphatidylserine from the extracellular monolayer to the cytosolic monolayer is inactivated. Together, this causes phosphatidylserine to rapidly accumulate at the cell surface.

How do the interiors of the ER, Golgi apparatus, endosomes, and lysosomes communicate with each other?

by small vesicles that bud off of one organelle and fuse with another FEEDBACK: Correct. In this way, transport vesicles carry soluble cargo proteins, as well as the proteins and lipids that are part of the vesicle membrane, from one organelle to another.

The shape of a cell and the mechanical properties of its plasma membrane are determined by a meshwork of fibrous proteins called what?

cell cortex FEEDBACK: Correct. This meshwork of protein filaments is attached to the underside of the plasma membrane.

The plasma membrane is involved in which activities?

cell growth and motility, cell recognition, cell signaling, import and export of nutrients and wastes FEEDBACK: Correct. The cell membrane is indeed involved in cell signaling and recognition, growth and motility, and the import of nutrients and export of wastes.The plasma membrane is not involved in DNA replication and repair or in the gene-silencing technique of RNA interference.

A genomic library is a collection of plasmids, each containing different chromosomal fragments from the genome of an organism. A cDNA library, on the other hand, contains only the ________ genome of an organism.

coding sequences of genes FEEDBACK: A cDNA library is a library of DNA fragments that are made from reverse transcription of mRNA transcripts. This type of library contains only the coding sequences of each gene, since introns are spliced out of mature mRNAs.

Most mitochondrial and chloroplast proteins are made within which part of the cell?

cytosol FEEDBACK: The synthesis of virtually all proteins in the cell begins on ribosomes in the cytosol. Although a few mitochondrial and chloroplast proteins are synthesized on ribosomes inside these organelles, most are made in the cytosol and subsequently imported. The proteins include a sorting signal that directs them to the correct intracellular location.Proteins moving from the cytosol into mitochondria or chloroplasts are transported across the organelle membrane by protein translocators located in the membrane. Unlike the transport through nuclear pores, the transported protein must usually unfold for the translocator to guide it across the hydrophobic interior of the membrane.

The first step of PCR requires that the DNA to be amplified is

denatured. FEEDBACK: In PCR, the target DNA must first be heated to denature the molecule into single-stranded DNA. This allows the primers to bind in the second step by base-pairing with their complementary sequence.

What can Sanger sequencing be used to do?

determine the nucleotide sequence of any purified DNA fragment FEEDBACK: Correct. Sanger, or dideoxy, sequencing can be used to determine the nucleotide sequence of any purified DNA fragment.

The movement of materials from the plasma membrane, through endosomes, and then to lysosomes describes which type of pathway?

endocytic pathway FEEDBACK: Vesicular transport between membrane-enclosed compartments of the endomembrane system is highly organized. The major outward secretory pathway starts with the synthesis of proteins on the ER membrane and their entry into the ER, and it leads through the Golgi apparatus to the cell surface—a process that is called exocytosis; a side branch of this pathway can carry materials from the Golgi through endosomes to lysosomes.Moving in the opposite direction, the major inward pathway carries materials from the plasma membrane, through endosomes, to lysosomes. This endocytic pathway is responsible for the ingestion and degradation of extracellular molecules.

The outer membrane of the nucleus is continuous with the membrane of which other organelle?

endoplasmic reticulum FEEDBACK: The nucleus, generally the most prominent organelle in eukaryotic cells, is surrounded by a double membrane known as the nuclear envelope. The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum (ER), a system of interconnected membranous sacs and tubes that often extends throughout most of the cell. The ER is the major site of synthesis of new membranes in the cell.The nuclear membranes and the membranes of the ER, Golgi apparatus, endosomes, and lysosomes most likely originated by invagination of the plasma membrane.

Intracellular condensates are non-membrane bound biochemical subcompartments that form due to phase separation among networks of weakly interacting molecules. Sabari et al., 2018, proposed that the transcriptional coactivator BRD4 helps form intracellular condensates containing other transcriptional proteins. A prediction of this proposal is that BRD4 should behave as a liquid within the condensate with rapid movement. Which procedure could be used to analyze movement of BRD2 in living cells?

fluorescence recovery after photobleaching (FRAP) FEEDBACK: Fluorescence recovery after photobleaching is a technique used to track the diffusion rate of fluorescently tagged molecules. The quicker the recovery of fluorescence in the bleached area, the faster the diffusion rate of the tagged molecules.

The CRISPR system can be used to activate a gene by altering the Cas9 enzyme in what way?

fusing an inactive Cas9 to a transcriptional activator FEEDBACK: Instead of targeting a Cas9 nuclease to a site for making a double-strand break for genome editing, CRISPR can be used to target various activities to specific locations in the genome. This is done by inactivating the Cas9 so that it is no longer a nuclease, and then attaching a protein with a desired activity.

You have the cDNA for a human gene that encodes a protein that can be used therapeutically. You want to produce this protein in yeast cells to provide a safe, reliable supply for patients. You have a yeast expression vector; this is a plasmid that contains a promoter followed by a multi-cloning site (MCS) where genes can be inserted. The MCS contains unique sites for both EcoRI and HindIII restriction enzymes. Your cDNA is flanked by EcoRI sites, so you cut the plasmid with EcoRI and ligate in your cDNA. Unfortunately, the cDNA can ligate in either direction, producing two different final constructs. Since you want the gene to be transcribed, the orientation relative to the promoter is important. To determine the orientation of the insert, you cut the plasmid with HindIII and determine fragment sizes. What technique can you use to determine the size of the fragments produced from the restriction digest of the plasmid?

gel electrophoresis FEEDBACK: Correct. Gel electrophoresis separates nucleic acids according to their size.

Which of the following libraries would be expected to be essentially the same?

genomic libraries made from mouse liver and kidney cells FEEDBACK: Correct. Genomic libraries produced from different cell types within the same organism should be the same, as the various cell types within a multicellular organism contain the same genomic DNA.

Which technique or reagent would be least likely to reveal whether a patient has been infected by a virus?

green fluorescent protein FEEDBACK: Correct. Green fluorescent protein is a "signal" molecule but does not have any inherent specificity. To detect a particular virus, it would need to be linked in some fashion to another molecule that would identify the presence of the virus.

Identify the component that associates with the Cas9 protein. Drag the correct RNA label to the target.

guide RNA FEEDBACK: Correct. The Cas9 protein along with a guide RNA designed by the experimenter are both artificially expressed in the cell or species of interest.

In the polymerase chain reaction, what is used to separate the two strands of a double-stranded DNA molecule?

high heat FEEDBACK: Correct. In PCR, high heat is used to separate the two strands of a double-stranded DNA molecule. The use of heating and cooling to separate the amplified double-stranded DNA molecules and then anneal the primers is what makes PCR such an efficient, rapid, and easily automated technique.

Which characteristic describes the tails of phospholipids?

hydrophobic FEEDBACK: Correct. The hydrocarbon tails of phospholipids tend to avoid contact with water, which helps drive the formation of the lipid bilayer.

Porin proteins—which form large, water-filled pores in mitochondrial and bacterial outer membranes—fold into β-barrel structures. The amino acids that face the outside of the barrel have what kind of side chains?

hydrophobic FEEDBACK: Correct. These hydrophobic side chains interact with the hydrophobic tails within the lipid bilayer. This arrangement allows the protein, which also contains hydrophilic amino acids and a hydrophilic peptide backbone, to penetrate the hydrophobic environment of the membrane.

Consider the following image of genes being highlighted on two homologous chromosomes and then answer the question regarding this experimental result. What technique was likely used to generate the experimental result that is shown above?

in situ hybridization FEEDBACK: Correct. In situ hybridization can be used to locate genes on isolated chromosomes. With in situ hybridization, investigators can visualize a specific nucleic acid sequence—either DNA or RNA— in its normal location within the cell.

In an electron transport chain, electrons are passed from one transmembrane electron carrier to another, driving proton movement across a membrane (see image below). The protons then flow through ATP synthase (not shown) to generate ATP. In a 2018 article (Budin, et al., Science vol. 362) researchers probed how membrane fluidity affects electron transport chain activity and ATP production in E. coli by manipulating membrane fluidity and measuring respiration. How could researchers have increased membrane fluidity?

increase the proportion of phospholipids with unsaturated fatty acids FEEDBACK: Unlike saturated fatty acids, unsaturated fatty acids contain kinks (see image below). These kinks prevent tight packing of adjacent phospholipids, thus increasing membrane fluidity.

Watch the animation on PCR, and then answer the questions. You have now added both PCR primers and successfully run the PCR, but you notice that the quantity of product generated is low. The graduate student you work with mentions that your expected product is quite long and that you may want to alter your PCR conditions to increase efficiency of product generation. Which of the following would help increase the amount of long PCR product generated?

increasing the elongation time when DNA polymerase synthesizes the new DNA FEEDBACK: Correct. Increasing the elongation time during DNA synthesis could allow more time to synthesize the longer strands of DNA and could increase product generation.

Which of the following terms describes a mouse in which a particular gene has been genetically eliminated?

knockout mouse FEEDBACK: Correct. "Knockout" is the term used to describe a mouse in which a particular gene has been genetically eliminated. In a knockout mouse, the activity of the gene is absent.

Gel electrophoresis separates DNA fragments from each other by virtue of which feature?

length FEEDBACK: Correct. Gel electrophoresis separates DNA fragments from each other by virtue of their length. Certain types of gels can differentiate between fragments that differ in length by a single nucleotide pair.

What is the starting material for making a cDNA library?

mRNA FEEDBACK: Correct. mRNA is the starting material, or the template, for making a cDNA library. The DNA that goes into a cDNA library is not genomic DNA; it is DNA copied from the mRNAs present in a particular type of cell. To prepare a cDNA library, all of the mRNAs are extracted, and double- stranded DNA copies of these mRNAs are produced.

What does the term "DNA cloning" refer to?

making many identical copies of a DNA molecule FEEDBACK: Correct. DNA cloning is the act of making many identical versions of a DNA molecule. When the cloning plasmid is inserted into a bacterial cell, the cell replicates (or clones) the plasmid many times.

What is a functionally specialized region of a cell membrane, typically characterized by the presence of specific proteins, called?

membrane domain FEEDBACK: Correct. Membrane domains are generated when cells restrict the movement of certain membrane proteins to localized areas within a cell membrane.

Watch the animation about lipids and the lipid bilayer, and then answer the questions. Imagine you collected bacteria from the sediment in a frozen lake in Minnesota in January and compared the membranes to membranes from bacteria collected from a lake in Texas in June. Consider how the membranes would likely differ.The membranes in bacteria from the Minnesota lake would most likely have which of the following?

more unsaturated lipid tails than membranes in Texas bacteria FEEDBACK: Correct. Unsaturated lipid tails with cis double bonds are kinked and pack less tightly than saturated lipids. The bacteria in a cold environment will have more of the unsaturated lipid tails to maintain fluidity even in cold temperatures.

Shown here is an expression vector. For the gene of interest to be expressed in the cells, the gene of interest must be cloned where in this vector?

next to the promoter, in the proper orientation FEEDBACK: Expression vectors contain a promoter that drives transcription of the gene of interest. Promoters have directionality, so for the gene to be expressed, the gene must be placed in the proper location relative to the promoter.

Which proteins bind to nuclear localization signals on newly synthesized proteins?

nuclear import receptors FEEDBACK: Correct. The nuclear localization signal on proteins destined for the nucleus is recognized by cytosolic proteins called nuclear import receptors. These receptors help direct a newly synthesized protein to a nuclear pore by interacting with the tentacle-like fibrils that extend from the rim of the pore into the cytosol.

Which of the following would produce the most fluid lipid bilayer?

phospholipids with tails of 18 carbon atoms and two double bonds FEEDBACK: Correct. A shorter chain length and double bonds both reduce the tendency of the phospholipid tails to interact with one another, thereby increasing the fluidity of the membrane.

DNA migrates through the pores of an agarose gel towards the ________ electrode; larger fragments travel more________.

positive; slowly FEEDBACK: DNA fragments can be separated by size by forcing their migration through a dense gel matrix; the larger fragments are slower to migrate because they are impeded by the matrix. The DNA fragments move toward the positive electrode due to their negative charge generated by the phosphate groups in the molecule's backbone.

What extra sequences are found in expression vectors that are not found in typical cloning vectors?

promoter sequences that direct the production of large quantities of mRNA FEEDBACK: Correct. Promoter sequences that direct the production of large quantities of mRNA are found in expression vectors but not in typical cloning vectors. The stronger the promoter, the more cellular RNA polymerase that transcribes the gene(s) of the vector.

Which feature of a cloning vector enables bacterial cells to replicate a plasmid to make many more copies?

replication origin FEEDBACK: For a piece of DNA to be replicated by the cell's machinery, it requires a replication origin, which is a sequence recognized by the cell for the cellular polymerase and other replication enzymes, in order to begin the copying process. Investigators take advantage of this phenomenon to isolate many copies of a desired plasmid.

In order to detect an infectious microorganism by PCR, what is required as a primer?

short sequences complementary to a part of the microbe's genome FEEDBACK: Correct. To detect an infectious microorganism using PCR, you would need short sequences complementary to a part of the microbe's genome. Primers that recognize any sequence in the microbial genome would permit PCR to amplify even a few copies of an infectious microbial genome. It is important that these target sequences are unique to the infectious microbe and do not also occur in the human genome.

DNA in a gel is most often visualized by

staining with a UV-reactive dye. FEEDBACK: DNA itself is colorless under visible light or blacklight. The DNA bands are visualized by adding a UV-reactive dye that interacts between bases of DNA molecules and observing under UV light.

Which of the following cannot be determined by comparing a nucleotide sequence to sequences available in public databases?

the gene's precise role in the physiology or development of the organism FEEDBACK: Correct. One cannot determine a gene's precise role in the physiology or development of an organism by comparing a nucleotide sequence to sequences available in public databases. Additional experiments are needed, such as examining when and where a gene is expressed or what happens to the cell or organism when that gene is deleted.

The chain-termination method of sequencing relies on the formation of partial copies of the DNA fragment that terminate at each position in the sequence. The fragments are separated by size and the terminating nucleotide is identified by

the position in the capillary and a unique fluorescent tag on each nucleotide. FEEDBACK: The chain-termination method of sequencing (or Sanger sequencing) amplifies the DNA of interest to form many molecules that are randomly terminated by the incorporation of one of four different fluorescently labeled dideoxynucleotides. The fragments are separated by size in a capillary gel matrix and the fluorescent tags are read to reveal the sequence of the DNA molecule.

In clone-by-clone sequencing, individual clones are first placed in order based on what factor?

the restriction sites they contain FEEDBACK: Correct. Before they are sequenced, individual clones are positioned on a physical map of the genome based on the pattern of restriction sites they contain. This restriction site "signature" can be used to order the fragments and place them on a known restriction map of the human genome.

In order to design primers for the polymerase chain reaction, what must be known about the DNA of interest?

the sequence at its ends FEEDBACK: Correct. PCR requires a set of primers that bracket the DNA of interest. As such, only the ends of the sequence of interest must be known so that the proper primers can be synthesized.

When a mixture of DNA fragments of different sizes is loaded into an agarose gel and a voltage is applied, the DNA will migrate in the gel. Which molecules will migrate the fastest?

the smallest fragments FEEDBACK: Correct. When a mixture of DNA fragments of different sizes is loaded onto an agarose gel and a voltage is applied, the smallest DNA fragment will migrate the fastest in the gel. The lowermost bands on the gel will contain the smallest DNA fragments. If the voltage is applied for too long, those fragments can actually run off the end of the gel.

Why must all living cells carefully regulate the fluidity of their membranes?

to permit membrane lipids and proteins to diffuse from their site of synthesis to other regions of the cell to allow membranes, under appropriate conditions, to fuse with one another and mix their molecules to ensure that membrane molecules are distributed evenly between daughter cells when a cell divides FEEDBACK: Correct. For all living cells, maintaining optimal membrane fluidity permits the diffusion of newly synthesized membrane lipids and proteins, ensures that membrane molecules are distributed evenly when a cell divides, and, under appropriate conditions, allows membranes to fuse with one another and mix their molecules.

Which of the following is a function of proteins in the plasma membrane?

transport molecules across the membrane, transmit extracellular signals to the cell interior, serve as anchors to attach the cell to the extracellular matrix, allow specific ions to cross the plasma membrane, thereby controlling its electrical properties FEEDBACK: Correct. Membrane proteins serve many functions. Some transport particular nutrients, metabolites, and ions across the lipid bilayer. Others anchor the membrane to macromolecules on either side. Still others function as receptors that detect chemical signals in the cell's environment and relay them into the cell interior, or work as enzymes to catalyze specific reactions at the membrane. Each type of cell membrane contains a different set of proteins, reflecting the specialized functions of the particular membrane.