Cell Bio

A researcher wants to determine whether a gene has a mutation, whether its mRNA expression has changed, and whether its protein is located in the nucleus. What should be analyzed for each question?

Genomic DNA for mutations (sequencing), RNA for expression (RT-qPCR), and protein for localization (immunofluorescence or subcellular fractionation).

How would overexpression, knockdown, and site-directed mutagenesis help determine a gene's function?

Overexpression increases the gene's output; knockdown reduces it; site-directed mutagenesis changes its DNA sequence. Comparing cellular responses reveals the gene's functional role.

A researcher suspects that a chromosome region has extra copies rather than a point mutation. Which technique should be used and why?

Array comparative genomic hybridization (aCGH), because it detects DNA copy-number variations across the genome.

Why would DNA sequencing be more appropriate than aCGH for identifying a specific nucleotide mutation?

Sequencing identifies changes in nucleotide sequence, whereas aCGH primarily detects gains or losses of DNA regions (copy-number variations).

Why can two complementary DNA strands separate when heated and reassociate when cooled?

Heat disrupts hydrogen bonds between complementary bases (denaturation). Cooling allows complementary sequences to base-pair again (renaturation or hybridization).

What are the three steps of PCR, and what occurs during each?

1. Denaturation: Heat separates DNA strands. 2. Annealing: Cooling allows primers to bind complementary sequences. 3. Extension: DNA polymerase extends the primers using dNTPs to synthesize DNA.

Why are two specific primers required to amplify a particular DNA region using PCR?

The primers bind complementary sequences flanking the target region on opposite strands, defining which DNA segment will be amplified.

Why does PCR use Taq polymerase instead of a typical eukaryotic DNA polymerase?

Taq polymerase comes from the thermophilic bacterium Thermus aquaticus and withstands repeated high-temperature denaturation, so it does not need to be replaced after every cycle.

If PCR begins with one double-stranded DNA molecule, how many copies are expected after n ideal amplification cycles, and why?

2^n copies, because each cycle approximately doubles the number of DNA molecules. Repeated cycles generate exponential amplification.

Why are short tandem repeats (STRs) useful for distinguishing individuals in forensic DNA analysis?

STR loci contain repeated DNA sequences whose repeat numbers vary greatly among individuals. These differences produce DNA fragments of different lengths after PCR.

Why can PCR amplification of one STR locus produce two different-sized DNA fragments from one individual?

The individual inherits one STR variant from each parent, and the maternal and paternal alleles may contain different numbers of repeats.

A crime-scene STR pattern matches a suspect at one locus. Why should additional loci be analyzed before identifying the suspect?

Unrelated people can share STR patterns at individual loci. Matching more independent loci greatly reduces the probability of a coincidental DNA fingerprint.

How is mRNA converted into double-stranded complementary DNA (cDNA)?

A poly(T) primer binds the mRNA poly(A) tail; reverse transcriptase synthesizes complementary DNA, the RNA is removed, and DNA polymerase synthesizes the second DNA strand.

Why can a genomic DNA clone contain sequences absent from a cDNA clone of the same gene?

Genomic DNA includes introns and other genomic sequences, whereas cDNA is produced from processed mRNA and represents the expressed, spliced sequence.

Why must reverse transcription occur before PCR amplification of an RNA virus such as SARS-CoV-2?

PCR amplifies DNA, not RNA. Reverse transcriptase first converts viral RNA into cDNA, which can then be amplified and detected.

A researcher wants to compare mRNA abundance between treated and untreated cells. Which PCR-based technique is appropriate, and what are its two steps?

RT-qPCR (Reverse Transcription Quantitative PCR): (1) Reverse transcriptase converts mRNA into cDNA. (2) Real-time qPCR amplifies and measures the cDNA to compare original mRNA levels.

How do reverse-transcription PCR and real-time quantitative PCR differ in purpose?

Reverse transcription converts RNA into cDNA for amplification; real-time quantitative PCR monitors DNA amplification to estimate the amount of starting target material.

How does SDS-PAGE allow proteins with different sizes and structures to be compared?

SDS denatures proteins and gives them a negative charge; the gel separates them primarily by size. Reducing agents can also break disulfide bonds.

Two proteins enter an SDS-PAGE gel, one large and one small. Which migrates farther toward the positive electrode, and why?

The smaller protein migrates farther because it passes more easily through the gel matrix, while larger proteins are restricted.

Why do DNA fragments migrate toward the positive electrode during agarose gel electrophoresis, and which fragments travel farthest?

DNA has a negatively charged phosphate backbone, so it moves toward the positive electrode. Smaller fragments migrate farther through the gel.

How are DNA fragments visualized after agarose gel electrophoresis in the lecture procedure?

Ethidium bromide intercalates into DNA and fluoresces under ultraviolet light, allowing separated DNA bands to be observed.

How do Coomassie staining and Western blotting differ in what they reveal about proteins?

Coomassie staining visualizes proteins in a gel generally, whereas Western blotting uses antibodies to identify specific proteins after transfer to a membrane.

What are the major steps of Western blotting, and why are primary and secondary antibodies used?

Separate proteins by SDS-PAGE, transfer them to a membrane, and detect them with antibodies. The primary antibody recognizes the target protein; the labeled secondary antibody enables detection.

A Western blot shows less target protein in a treated sample but equally intense GAPDH bands in all samples. What does this suggest?

The target protein is reduced in the treated sample, and comparable GAPDH loading-control bands support that the difference is not simply due to unequal protein loading.

Why is a loading control necessary when comparing protein expression across Western blot samples?

It provides a reference for relative protein loading, helping distinguish genuine expression differences from differences in how much protein was loaded.

An antibody recognizes several bands of different molecular weights corresponding to the same gene's protein products. What could explain this pattern?

Different splice variants can produce protein products of different sizes, as illustrated by the EVI1 Western blot in the lecture.

How can antibody-based fluorescence microscopy be used to determine where a protein is located inside a cell?

Fix cells, block nonspecific binding, incubate with antibodies against the target protein, and visualize fluorescence. The staining pattern identifies the protein's cellular location.

Why are formaldehyde, goat serum, and Triton X-100 used during the lecture's immunofluorescence procedure?

Formaldehyde fixes cellular structures, goat serum blocks nonspecific antibody binding, and Triton X-100 helps antibodies access intracellular proteins.

A researcher wants to determine whether a protein is nuclear or cytoplasmic rather than simply measure its total abundance. Which methods are appropriate?

Immunofluorescence microscopy or subcellular fractionation. Western blotting of whole-cell lysate measures protein abundance but does not directly reveal its location.

Why is reverse-phase protein array (RPPA) useful for analyzing large numbers of protein samples?

RPPA is a highly sensitive, quantitative, high-throughput method using validated antibodies. It can analyze many protein samples with very little starting material.

How do restriction nucleases allow researchers to isolate specific DNA fragments from a genome?

They recognize particular DNA sequences, typically 4–8 base pairs long, and cut DNA at those sites, producing fragments that can be separated or cloned.

What determines whether a restriction enzyme produces sticky ends or blunt ends?

The arrangement of the DNA cuts: staggered cuts produce single-stranded overhangs (sticky ends), while cuts directly across both strands produce blunt ends.

A linear DNA molecule has four restriction sites, all recognized by the same enzyme. How many fragments result from complete digestion?

Five fragments. For linear DNA, the number of fragments equals the number of cuts plus one.

A circular plasmid has four restriction sites recognized by an enzyme. How many fragments result from complete digestion, and why?

Four fragments. For circular DNA with at least one cut, the number of fragments equals the number of cuts.

A linear DNA molecule has two EcoRI sites and two HindIII sites. How many fragments result from EcoRI alone, HindIII alone, and both enzymes together?

EcoRI alone: 3 fragments. HindIII alone: 3 fragments. Both enzymes: 5 fragments, assuming all four sites are distinct and fully cut.

A circular plasmid contains three EcoRI sites, one HindIII site, and one PstI site. How many fragments result from EcoRI alone, HindIII alone, EcoRI + HindIII, and all three enzymes?

EcoRI: 3; HindIII: 1; EcoRI + HindIII: 4; all three: 5. Each distinct cut on circular DNA produces one fragment.

A 4000-bp linear DNA molecule has an EcoRI site at 1000 bp and HindIII sites at 3000 and 3800 bp. What fragment sizes result after digestion with both enzymes?

1000 bp, 2000 bp, 800 bp, and 200 bp. The fragments correspond to distances between consecutive cuts and the DNA ends.

Why do complementary sticky ends facilitate recombinant DNA formation, and what is DNA ligase's role?

Complementary overhangs can base-pair, positioning fragments together. DNA ligase forms covalent bonds in the sugar-phosphate backbone, permanently joining them.

How do Southern and Northern blotting differ, and what principle do both techniques use?

Southern blotting analyzes DNA; Northern blotting analyzes RNA. Both use complementary nucleic acid hybridization to detect particular sequences.

What is the overall process for cloning a DNA sequence in bacteria?

Cut DNA and a vector with restriction enzymes, ligate the desired fragment into the plasmid, introduce the recombinant plasmid into bacteria, grow the bacteria, and recover amplified DNA.

A DNA fragment and plasmid have compatible ends, but DNA ligase is omitted. What happens?

The fragments may temporarily base-pair through complementary sticky ends, but a stable covalently joined recombinant DNA molecule will not form efficiently.

What are the three key plasmid-vector elements emphasized in the lecture, and what does each do?

Promoter: drives transcription; origin of replication: allows DNA replication in the host; antibiotic-selection marker: allows identification of bacteria carrying the plasmid.

Why are antibiotic-selection markers useful after introducing recombinant plasmids into bacterial cells?

They allow bacteria containing the plasmid to survive under the appropriate antibiotic-selection conditions, distinguishing them from cells that did not acquire the vector.

A researcher clones a gene into an expression vector with a strong promoter. What is the intended result?

Increased transcription of the inserted gene, allowing the host cells to produce large amounts of the encoded protein.

How can fusion of a target gene to green fluorescent protein (GFP) help researchers study the target protein?

The target gene is fused to GFP DNA, producing a fluorescent chimeric protein that can be visualized in cells to study expression or localization.

A researcher changes one specific nucleotide in a cloned gene and observes altered protein function. Which technique was used, and what does the experiment reveal?

Site-directed mutagenesis. The altered functional response helps determine the importance of that nucleotide or the resulting amino acid change.

What is signal transduction, and how does an extracellular signal produce an intracellular response?

Signal transduction converts information from an extracellular signal into intracellular signaling events. Ligand binding activates a receptor, which relays information through signaling molecules to effector proteins.

How do endocrine, paracrine, and autocrine signaling differ?

Endocrine: hormones travel through blood to distant cells. Paracrine: local mediators act on nearby cells. Autocrine: a cell responds to a signaling molecule it produces itself.

How do neuronal and contact-dependent signaling differ from endocrine signaling?

Neuronal signaling transmits directed signals over potentially long distances; contact-dependent signaling requires direct contact between cells. Endocrine signaling distributes hormones through the bloodstream.

Why do large hydrophilic ligands generally require cell-surface receptors, while small hydrophobic ligands can use intracellular receptors?

Large or hydrophilic molecules cannot readily cross the lipid bilayer. Small hydrophobic molecules can cross and bind receptors in the cytosol or nucleus.

EGF and cortisol act on cells through different receptor locations. Which receptor class does each use, and why?

EGF is a large protein ligand that binds cell-surface receptors. Cortisol is a small hydrophobic steroid that crosses the membrane and binds intracellular receptors.

How can the same signaling molecule produce different responses in different target cells?

Different cell types express different receptors and intracellular signaling proteins, so the same ligand can activate different effector pathways and cellular responses.

Acetylcholine slows pacemaker activity in one cell, stimulates secretion in another, and causes contraction in skeletal muscle. Why are these effects different?

Each target cell has a different receptor and intracellular signaling system, causing acetylcholine to activate distinct effector proteins and biological responses.

Why might a cell survive with one combination of extracellular signals but divide or differentiate when additional signals are present?

Cells integrate multiple extracellular signals. Different combinations activate different intracellular pathways, producing distinct outcomes such as survival, proliferation, differentiation, or death.

Why can acetylcholine trigger a rapid response while a growth-factor signal may require much longer?

Rapid responses modify the activity of pre-existing proteins; slower responses require changes in gene expression and synthesis of new proteins.

What properties of ligand-receptor interactions allow selective but reversible cellular responses?

Ligands bind specific receptors through multiple weak interactions, causing receptor conformational changes. Reversible binding allows the signal to be regulated or terminated.

What are the five functions of intracellular signaling proteins summarized by RAIDM?

Relay signals, Amplify signals, Integrate information from multiple pathways, Distribute signals to multiple effectors, and Modulate upstream components through feedback.

How do positive and negative feedback affect signaling pathways differently?

Positive feedback reinforces pathway activation and can create a strong response. Negative feedback inhibits earlier components, limiting signaling and potentially producing oscillations.

Why are second messengers effective at amplifying extracellular signals, and what are the examples emphasized in the lectures?

They are small intracellular molecules that can spread a signal to many downstream targets, often activating kinases. Examples: cAMP, IP3, DAG, and Ca²⁺.

How do protein kinases and protein phosphatases regulate molecular switches in signaling pathways?

Kinases add phosphate groups to proteins, while phosphatases remove them. Phosphorylation changes protein activity and can turn a signaling component on or off.

How do GEFs and GAPs control GTP-binding molecular switches?

GEFs promote GDP release and GTP binding, activating the protein. GAPs stimulate GTP hydrolysis to GDP, inactivating it.

What are the six overall stages of signal transduction emphasized in the lecture?

Synthesis of the ligand → release → transport → receptor detection → intracellular signaling and cellular response → removal or termination of the signal.

What are the three major classes of cell-surface receptors, and how does each transmit signals?

Ion-channel-coupled receptors open or close ion channels; GPCRs activate G proteins; enzyme-linked receptors activate catalytic domains or associated enzymes.

How does the structure of a GPCR differ from that of a typical receptor tyrosine kinase (RTK)?

A GPCR is a single polypeptide crossing the plasma membrane seven times and coupling to G proteins. An RTK generally has one membrane-spanning segment and intracellular tyrosine kinase activity.

What are the three subunits of a heterotrimeric G protein, and which can regulate downstream effectors?

Alpha (α), beta (β), and gamma (γ). Activated α-GTP and the βγ complex can regulate target proteins such as enzymes or ion channels.

What sequence of events activates a heterotrimeric G protein after a ligand binds its GPCR?

The activated receptor promotes GDP release from Gα and GTP binding. Gα-GTP separates from Gβγ, allowing the activated components to regulate target proteins.

How does a heterotrimeric G protein turn itself off after receptor activation?

The α subunit hydrolyzes GTP to GDP through its intrinsic GTPase activity, disengages from its effector, and reassociates with βγ to restore the inactive complex.

How do stimulatory Gs and inhibitory Gi proteins differ in their effects on second-messenger production?

Gs stimulates downstream enzyme activity, while Gi inhibits it; for adenylyl cyclase, Gs increases cAMP production and Gi decreases it.

Acetylcholine activates a Gi-coupled receptor in a heart pacemaker cell. How does this slow the heart rate?

Activated Gβγ opens K⁺ channels, increasing K⁺ permeability and making the membrane harder to electrically activate, which slows pacemaker firing.

If the G protein α subunit could not hydrolyze GTP, what would happen to its downstream signaling?

The α subunit would remain active longer, prolonging effector activation or inhibition because the normal GTPase-dependent shutoff is impaired.

What reaction does adenylyl cyclase catalyze, and how does the product transmit the signal?

Adenylyl cyclase converts ATP into cAMP. cAMP serves as a second messenger that activates protein kinase A (PKA).

How does cAMP activate protein kinase A (PKA), and what types of proteins can PKA affect?

cAMP binds PKA regulatory subunits, releasing active catalytic subunits. PKA phosphorylates serine/threonine residues on enzymes and proteins controlling gene expression.

If phosphodiesterase (PDE) activity increases, what happens to cAMP signaling and why?

cAMP signaling decreases because PDE breaks down cAMP into AMP, terminating second-messenger stimulation of PKA.

How does the cytosolic PKA pathway allow epinephrine to stimulate glycogen breakdown?

Epinephrine activates GPCR → Gs → adenylyl cyclase → cAMP → PKA. PKA activates the phosphorylation cascade that stimulates glycogen phosphorylase and glycogen breakdown.

How can the same cAMP-PKA pathway produce a slower change in gene expression rather than an immediate metabolic response?

Activated PKA can enter the nucleus and phosphorylate transcription regulators, activating target-gene transcription instead of only modifying cytosolic enzymes.

What reaction does phospholipase C (PLC) catalyze, and what happens to its two products?

PLC cleaves membrane PIP2 into IP3 and DAG. IP3 promotes Ca²⁺ release from the ER, while membrane-associated DAG helps activate protein kinase C (PKC).

How does IP3 increase cytosolic Ca²⁺ in the inositol phospholipid signaling pathway?

IP3 diffuses to the endoplasmic reticulum and binds IP3-gated Ca²⁺ channels, opening them and releasing stored Ca²⁺ into the cytosol.

Why is DAG able to activate a membrane-associated kinase while IP3 acts on the endoplasmic reticulum?

DAG remains associated with the plasma membrane and participates in PKC activation; IP3 is soluble and diffuses through the cytosol to ER Ca²⁺ channels.

If phospholipase C is inhibited after activation of a Gq-coupled receptor, which downstream responses are reduced?

PIP2 cleavage decreases, reducing IP3 and DAG formation. Consequently, IP3-mediated Ca²⁺ release and DAG-dependent PKC activation decrease.

How do tyrosine kinase receptors, serine/threonine kinase receptors, and enzyme-linked receptors without intrinsic kinase activity differ?

They use different catalytic mechanisms: some phosphorylate tyrosine residues, some phosphorylate serine/threonine residues, and others recruit separate intracellular proteins with kinase activity.

What sequence of events activates a typical receptor tyrosine kinase (RTK)?

Ligand binding promotes receptor dimerization → intracellular kinase domains activate → receptors autophosphorylate tyrosines → signaling proteins bind and initiate downstream pathways.

Why are phosphorylated tyrosines on activated RTKs important beyond simply activating the receptor?

They create docking sites for intracellular signaling proteins, including adaptor proteins with SH2 domains, enabling assembly of signaling complexes.

An RTK can still bind its ligand but cannot phosphorylate its cytoplasmic tyrosines. What downstream effect would you expect?

Recruitment of phosphotyrosine-binding signaling proteins would be impaired, reducing activation of downstream pathways despite ligand binding.

What mechanisms can terminate signaling from an activated receptor tyrosine kinase?

Protein phosphatases remove activating phosphates, while receptor endocytosis followed by degradation in lysosomes can reduce the amount of active receptor.

How does an activated RTK convert inactive Ras-GDP into active Ras-GTP?

Activated RTK recruits adaptor proteins and the Ras-GEF SOS. SOS promotes GDP release and GTP binding, activating membrane-associated Ras.

Why is Ras classified as a monomeric GTPase rather than a heterotrimeric G protein?

Ras is a single small GTP-binding protein attached to the cytoplasmic face of the membrane, whereas heterotrimeric G proteins contain α, β, and γ subunits.

What happens if Ras is unable to hydrolyze GTP or its GAP-mediated inactivation is disrupted?

Ras remains GTP-bound and active for longer, sustaining downstream kinase signaling rather than returning normally to its inactive GDP-bound state.

How does activated Ras transmit signals through the MAP kinase pathway?

Ras-GTP activates MAPKKK → MAPKK → MAPK through a phosphorylation cascade. Activated MAPK phosphorylates effector proteins, altering protein activity or gene expression.

A drug blocks MAPKK activity even though Ras remains activated. What happens to the downstream MAP kinase pathway?

MAPK activation decreases because MAPKK can no longer efficiently phosphorylate MAPK, interrupting transmission of the signal to downstream effectors.

How does PI3-kinase (PI3K) activation create new binding sites in the plasma membrane?

PI3K phosphorylates membrane inositol phospholipids, producing phosphorylated lipid docking sites such as PIP3 that recruit specific intracellular signaling proteins.

How does activated PI3K lead to activation of AKT (protein kinase B)?

PI3K generates phosphorylated membrane lipids that recruit signaling proteins and AKT to the membrane, allowing activation of AKT by upstream protein kinases.

How does AKT promote survival by targeting BAD?

AKT phosphorylates and inactivates pro-apoptotic BAD, allowing Bcl-2-mediated inhibition of apoptosis and increasing cell survival.

How does AKT promote cell growth through TOR, and what is the role of rapamycin?

AKT activates the TOR pathway, increasing protein synthesis and decreasing protein degradation. Rapamycin inhibits TOR and reduces these growth-promoting effects.

What happens to AKT-mediated cell survival and growth if PI3K signaling is inhibited?

↓ PI3K activity → ↓ PIP₃ → ↓ AKT docking & activation → ↓ BAD inhibition & ↓ TOR signaling → weakened cell survival and growth.

How do the products of adenylyl cyclase, PLC, and PI3K differ?

Adenylyl cyclase produces cAMP; PLC cleaves PIP2 into IP3 and DAG; PI3K phosphorylates membrane inositol phospholipids to create docking sites. These initiate different downstream signaling mechanisms.

How does Notch signaling transmit information directly from a neighboring cell to the nucleus?

Membrane-bound Delta on a neighboring cell binds Notch, causing Notch cleavage. Its released intracellular tail enters the nucleus and activates Notch-responsive genes.

Why is Notch signaling considered more direct than typical GPCR or RTK signaling?

Notch releases an intracellular receptor fragment that directly participates in regulating transcription, rather than relying primarily on extended G-protein or kinase cascades.

Why can GPCR and RTK pathways influence the same cellular response despite using different receptors?

Their downstream kinase networks can converge on common transcription regulators and effector proteins, integrating multiple signals to control cell behavior.

A cell receives one signal that activates a kinase and another that inhibits it. Why can the final response not be predicted from either signal alone?

Intracellular pathways integrate positive and negative inputs. The resulting response depends on their combined effects on shared signaling proteins and downstream targets.

What are the major ways a cell turns off an intracellular signaling response?

Remove or metabolize the ligand, sequester or degrade receptors, break down second messengers, alter target-protein phosphorylation through phosphatases, and inactivate GTP-binding switches through GTP hydrolysis.