Final Exam Cell and Molec

Question 1

What are the major checkpoints in the cell cycle?

Answer 1

G1/S checkpoint (restriction point), G2/M checkpoint, spindle assembly checkpoint (M)

Question 2

What is the function of the G2/M checkpoint?

Answer 2

It ensures DNA replication is complete and undamaged.

Question 4

What is the function of the spindle assembly checkpoint (M)?

Answer 4

It ensures chromosomes are properly attached to the spindle before anaphase.

Question 5

What is the function of the G1/S checkpoint?

Answer 5

It checks for DNA damage and sufficient nutrients.

Question 6

What is anaphase?

Answer 6

A stage in cell division where replicated chromosomes, or sister chromatids, separate and move to opposite ends of the cell.

Question 7

What proteins regulate the cell cycle?

Answer 7

Cyclins and cyclin-dependent kinases (CDKs). Cyclins fluctuate in concentration; CDKs are activated by binding specific cyclins.

Question 8

What is transcriptional regulation?

Answer 8

Control of when and how much mRNA is made from DNA. Involves transcription factors, enhancers/silencers, epigenetic marks (methylation, acetylation).

Question 9

What is translational regulation?

Answer 9

Control of when and how much protein is made from the mRNA. Affected by RNA secondary structures, binding proteins, and initiation sequence.

Question 10

What are Kozak’s rules?

Answer 10

Define the optimal sequence for translation initiation in eukaryotes: (gcc)gccRccAUGG. The purine (R) at -3 and G at +4 are most critical.

Question 11

What are oncogenes?

Answer 11

Mutated proto-oncogenes that promote uncontrolled cell growth (e.g., Ras, Myc). Dominant mutations.

Question 12

What are tumor suppressor genes?

Answer 12

Genes that inhibit cell cycle or promote apoptosis (e.g., p53, Rb). Mutations are typically recessive.

Question 13

What is apoptosis and its role in cancer?

Answer 13

Programmed cell death; removes damaged or unneeded cells. Failure of apoptosis contributes to tumor development.

Question 14

How does the immune system combat cancer?

Answer 14

Cytotoxic T cells recognize and kill abnormal cells expressing tumor antigens. Cancer cells may evade detection via immune checkpoints.

Question 15

What are the key properties of stem cells?

Answer 15

Self-renewal: Ability to divide and remain undifferentiated. Potency: Ability to differentiate into multiple cell types (totipotent, pluripotent, multipotent, unipotent).

Question 16

What are induced pluripotent stem cells?

Answer 16

Differentiated cells reprogrammed to pluripotency by introducing transcription factors.

Question 17

What are suppressor screens used for?

Answer 17

Identify genes that compensate for or rescue the phenotype of a mutant. Helps identify interacting genes or pathways.

Question 18

What is RNA interference (RNAi)?

Answer 18

A process where double-stranded RNA induces degradation of specific mRNA, silencing gene expression.

Question 19

What are HATs and what is their role?

Answer 19

Histone acetyltransferases add acetyl groups to histone tails, loosening chromatin and promoting transcription.

Question 20

What were key findings in the Tat/HAT paper?

Answer 20

Tat was shown to recruit HATs (e.g., p300, CBP), promoting transcription of HIV genes. Demonstrated interplay between viral protein and host chromatin machinery.

Question 21

What is Southern blotting used for?

Answer 21

Detects specific DNA sequences using labeled DNA probes. Useful for genotyping, mutation detection, and gene mapping.

Question 22

What is Northern blotting used for?

Answer 22

Detects RNA expression. Assesses mRNA size and abundance, useful for studying gene expression regulation.

Question 23

What is Western blotting used for?

Answer 23

Detects proteins using antibodies. Provides information on protein size, expression levels, and post-translational modifications.

Question 24

Give an overview of how Southern blotting is performed.

Answer 24

1. DNA extraction: isolate DNA from cells or tissues.
2. Restriction Enzyme Digestion: Cut DNA with restriction enzymes into fragments.
3. Gel Electrophoresis: Run digested DNA on an agarose gel. Separates the DNA based on size (smaller fragments move further).
4. Denaturation (in-gel): Soak the gel in an alkaline solution (e.g., NaOH) to denature DNA into single strands.
5. Blotting (transfer): Transfer single stranded DNA to a membrane.
6. Hybridization: Incubate the membrane with a labeled DNA probe (radioactive, fluorescent, or chemiluminescent) complementary to the target sequence.
7. Washing: Remove excess, unbound probe.
8. Use autoradiography or imaging systems to detect probe binding. Dark bands indicate presence and size of the target DNA.

Question 25

Give an overview of how Northern blotting is performed.

Answer 25

Goal: measure DNA expression levels and sizes. 1. RNA extraction: isolate total RNA or mRNA. 2. Denaturing gel electrophoresis: Use formaldehyde-agarose gel to prevent RNA secondary structures. Separates RNA by size. 3. Transfer (blotting): Transfer RNA to a membrane. 4. Crosslinking: UV light or baking is used to fix RNA onto the membrane. 5. Hybridization: Incubate with a labeled DNA or RNA probe complementary to the target mRNA. 6. Washing: Remove excess probe. 7. Detection: Visualize signal using autoradiography or chemiluminescence. Band intensity reflects RNA abundance.

Question 26

Give an overview of how Western blotting is performed.

Answer 26

Goal: Detect and quantify specific proteins using antibodies. 1. Protein extraction: Lyse cells and extract total protein. 2. SDS-PAGE electrophoresis: proteins are denatured with SDS and separated by size on a polyacrylamide gel. 3. Transfer to membrane: Use electroblotting to transfer proteins from the gel to a PVDF or nitrocellulose membrane. 4. Blocking: Incubate membrane in blocking buffer to prevent non-specific antibody binding. 5. Primary Antibody Incubation: Add an antibody that specifically binds the target protein. 6. Secondary Antibody Incubation: Add a labeled secondary antibody (enzyme-linked) that binds the primary antibody. 7. Detection: Add a substrate for the enzyme that produced a chemiluminescent or colorimetric signal. Visualize with film or imaging system.

Question 27

What are the stages of the cell cycle?

Answer 27

G1 → S → G2 → M, with G0 as a resting state.

Question 28

How is the cell cycle regulated?

Answer 28

By cyclins/CDKs.

Question 29

How are these stages 'turned on'?

Answer 29

Cyclin expression is transcriptionally regulated and degraded by the ubiquitin-proteasome system. Activation often depends on external signals like growth factors (via MAPK pathway).

Question 30

What is a suppressor screen?

Answer 30

A genetic technique to find mutations that rescue the phenotype of an existing mutant, revealing interacting pathways or redundant functions.

Question 31

What is an example of a suppressor mutation?

Answer 31

A mutation in a repressor gene that restores expression of a gene silenced in the original mutant.

Question 32

What is RNA interference (RNAi)?

Answer 32

A post-transcriptional gene silencing mechanism where dsRNA triggers degradation of homologous mRNA, often used in loss-of-function studies.

Question 33

What are types of post-transcriptional regulation?

Answer 33

Alternative splicing, RNA editing, mRNA stability, microRNAs, RNA localization, Translation efficiency.

Question 34

What is the Shine-Dalgarno sequence?

Answer 34

A ribosome-binding site upstream of AUG in prokaryotic mRNA: AGGAGG, pairs with 16S rRNA.

Question 35

What is the Kozak sequence?

Answer 35

Eukaryotic translation start consensus: gccRccAUGG, where R is a purine at -3 and G at +4 enhances initiation.

Question 36

How do signaling pathways affect the cell cycle and cancer?

Answer 36

MAPK pathway: Stimulates proliferation (often hyperactive in cancers). PI3K/AKT: Promotes survival and growth; can activate mTOR. Wnt/β-catenin: Promotes stemness and proliferation. p53 pathway: Suppresses tumors by inducing cell cycle arrest or apoptosis.

Question 37

What is the difference between potency and plasticity?

Answer 37

Potency: Cell’s intrinsic ability to differentiate (totipotent > pluripotent > multipotent). Plasticity: Ability of a differentiated cell to reprogram into a different cell type.

Question 38

What are general features of stem cells?

Answer 38

Self-renewal, Potency, Undifferentiated chromatin (open euchromatin), Expression of pluripotency factors (Oct4, Sox2, Nanog).

Question 39

How is chromatin structure in stem cells different?

Answer 39

Loosely packed (euchromatin-rich), High histone acetylation, low DNA methylation, Poised for transcription of many lineage genes.

Question 40

How do cancer cells increase potency/plasticity?

Answer 40

Reactivate stem cell genes, acquire epigenetic flexibility, and enhance transdifferentiation potential (e.g., via Wnt, Myc, Oct4 reactivation).

Question 41

How does epigenetics apply to stem cells and cancer?

Answer 41

Epigenetic marks regulate stemness and differentiation. Cancer cells often reprogram their epigenome to gain plasticity and silence tumor suppressors.

Question 42

What are microRNAs and their role?

Answer 42

Small non-coding RNAs (~22 nt) that bind 3' UTRs of mRNAs and repress translation or cause degradation.

Question 43

How do miRNAs relate to cancer?

Answer 43

OncomiRs: Promote cancer by silencing tumor suppressors. Tumor suppressor miRNAs: Lost in cancer, leading to oncogene overexpression.

Question 44

What is RNA splicing?

Answer 44

Removal of introns from pre-mRNA and joining of exons to form mature mRNA.

Question 45

What is alternative splicing?

Answer 45

Process that allows a single gene to produce multiple mRNA isoforms by combining exons in different ways.

Question 46

Why is alternative splicing important?

Answer 46

Increases proteome diversity. Errors can lead to diseases, including cancer and developmental disorders.

Question 47

What is post-transcriptional processing?

Answer 47

Post-transcriptional processing refers to the modifications made to pre-mRNA (the initial RNA copy of a gene) after transcription but before translation in eukaryotes. These steps prepare the mRNA for export from the nucleus, protect it from degradation, and regulate its translation efficiency.

Question 48

What are the major post-transcriptional processing events?

Answer 48

1. 5' Capping 2. 3' Polyadenylation 3. RNA splicing 4. RNA editing 5. RNA export

Question 49

What affects mRNA expression (and transcription)?

Answer 49

1. Transcription factor binding 2. Chromatin structure 3. Epigenetic modifications 4. Non-coding RNAs

Question 50

What is the role of the 5' cap on mRNA?

Answer 50

It protects the mRNA from 5' to 3' exonucleases, it is required for ribosome recognition, and it interacts with a key translation initiation factor.

Question 51

What happens when the 5' cap is removed from mRNA?

Answer 51

Ribosomes cannot efficiently bind to the mRNA which causes translation to be drastically reduced/blocked. The mRNA becomes unstable and rapidly degraded by exonucleases. Translation may only proceed via cap-independent mechanisms, which are more rare.

Question 52

What is the purpose of the polyA tail in mRNA?

Answer 52

It enhances mRNA stability, promotes efficient translation initiation, and interacts with a poly-A binding protein, which loops the mRNA and helps recruit the ribosome.

Question 53

What happens if the polyA tail is removed (deadenylation)?

Answer 53

The mRNA becomes unstable and rapidly degrades from the 3' end. Translation is less efficient, since the closed-loop formation is disrupted. Deadenylation is often the first step in mRNA decay.

Question 54

Where does most translation occur?

Answer 54

Translation predominantly occurs on ribosomes. In eukaryotic cells, translation mainly happens in the cytoplasm on free floating ribosomes or on ribosomes that are attached to the rough ER.

Question 55

What causes removal of introns?

Answer 55

RNA splicing removes introns from pre-mRNA and joins exons to form mature mRNA. This process is carried out by a molecular machine called the spliceosome.

Question 56

Why is splicing important?

Answer 56

It ensures accurate gene expression and enables alternative splicing, increasing protein diversity.

Question 57

Does the reading frame need to be correct when splicing? If so, why?

Answer 57

Yes, the reading frame must be properly maintained to produce a functional protein. If the reading frame is not correct, the incorrect amino acids will be coded for and a nonfunctional or truncated protein will result due to premature stop codons.

Question 58

Is it possible for different spliceosomes to give different splicing patterns?

Answer 58

Yes, different spliceosomes can recognize different boundaries on the pre-mRNA. This leads to different combinations of exons being joined together, resulting in multiple versions of mature mRNA from a single gene.

Question 59

Why do different cells have different spliceosomes?

Answer 59

All cells have fundamental splicing machinery but the context in which the machinery operates is different. This is a crucial mechanism for generating the vast complexity and functional specialization of different cells within a multicellular organism, allowing a limited number of genes to produce a much larger variety of proteins.

Question 60

What are snRNAs?

Answer 60

Small nuclear RNAs (snRNAs) are a class of small RNA molecules, typically around 150 nucleotides long, found in the nucleus of eukaryotic cells. They are crucial components of the spliceosome, the large molecular machine responsible for RNA splicing. They are essentially the functional RNA parts of the spliceosome. They associate with specific proteins to form small nuclear ribonucleoproteins (snRNPs) which are the building blocks of the spliceosome.