I.

The Human Genome and Gene Regulation

1. Describe the structure and functional organization of the human genome, highlighting the relative
contribution of coding and noncoding regions.

The human genome comprises approximately 3.2 billion base pairs distributed across 23 pairs of
chromosomes and the mitochondrial genome. The genome is organized into coding and noncoding regions,
each serving distinct but interrelated functions in maintaining cellular structure, function, and regulation.

Coding Regions

● Protein-coding sequences, known as exons, represent only about 1.5% of the genome. These
regions contain the instructions for synthesizing proteins, which perform structural, enzymatic, and
regulatory functions within the cell.
● These exonic sequences are interspersed with introns, which are noncoding segments within genes
that are transcribed but spliced out during mRNA processing.
● The total number of protein-coding genes is approximately 20,000 to 25,000, yet the diversity of the
proteome far exceeds this number due to mechanisms such as alternative splicing, post-
translational modifications, and gene recombination.

Noncoding Regions

While the protein-coding portion of the genome is limited, over 98.5% of the genome is composed of
noncoding DNA, which plays crucial roles in gene regulation and chromosomal integrity. These regions
include:

1. Regulatory Sequences
○ Promoters, enhancers, silencers, and insulators regulate gene transcription.
○ These elements interact with transcription factors and chromatin modifiers to control gene
expression spatially and temporally.

2. Noncoding RNAs (ncRNAs)

○ These RNAs are transcribed but not translated into protein. Key types include:
■ MicroRNAs (miRNAs): Regulate gene expression post-transcriptionally by binding to
target mRNAs and promoting their degradation or inhibiting translation.
■ Long noncoding RNAs (lncRNAs): Involved in chromatin remodeling, transcriptional
regulation, and scaffolding of nuclear structures.

3. Structural DNA Elements

○ Centromeres and telomeres maintain chromosome integrity during cell division.
○ Origins of replication ensure proper DNA duplication.

4. Repetitive Elements
○ Includes transposable elements such as LINEs (long interspersed nuclear elements) and SINEs
(short interspersed nuclear elements), which make up a substantial portion of the genome.
○ These elements may influence gene expression and genome evolution but can also contribute
to genomic instability.
5. Pseudogenes
○ These are gene-like sequences that have lost their coding potential due to mutations. While
traditionally thought to be nonfunctional, some pseudogenes may have regulatory roles.
2. Differentiate between protein-coding DNA and noncoding DNA, and explain the regulatory
functions of noncoding elements such as enhancers, silencers, and insulators.

The human genome is organized into two fundamental categories based on function: protein-coding DNA
and noncoding DNA. Though protein-coding sequences are responsible for the synthesis of proteins, the
majority of the genome consists of noncoding sequences that play crucial regulatory and structural roles.
 Protein-Coding DNA

● Definition: These are genomic regions that are transcribed and translated into proteins.
● Structure: Composed of exons within genes, which contain codons that specify amino acids.
● Proportion of Genome: Accounts for approximately 1.5% of the entire human genome.
● Function: Directs the synthesis of polypeptides, which fold into functional proteins involved in structural
integrity, enzymatic activity, signaling, transport, and cellular regulation.
● Transcription and Translation: Protein-coding genes are transcribed into messenger RNA (mRNA),
which undergoes processing (e.g., splicing, capping, polyadenylation) before being translated by
ribosomes into proteins.

Noncoding DNA

● Definition: These regions are not translated into proteins but serve regulatory, structural, or
evolutionary roles.
● Structure: Includes regulatory sequences, noncoding RNAs (e.g., miRNAs, lncRNAs), introns,
repetitive elements, pseudogenes, centromeres, and telomeres.
● Proportion of Genome: Constitutes over 98.5% of the genome.
● Function: Regulates gene expression at the transcriptional and post-transcriptional levels, maintains
chromosomal stability, and contributes to nuclear organization.

Regulatory Functions of Noncoding Elements

Among the most crucial noncoding elements in gene regulation are enhancers, silencers, and insulators.
These elements interact with transcription factors, chromatin-modifying proteins, and the transcriptional
machinery to control the timing, location, and magnitude of gene expression.

Enhancers

● Definition: DNA sequences that increase transcriptional activity of associated genes.

● Location: May be located upstream, downstream, or within introns, and can act over long genomic
distances.
● Function: Serve as binding sites for activator transcription factors. Upon binding, enhancers interact
with promoters via DNA looping, thereby facilitating recruitment of RNA polymerase II and coactivator
complexes.
● Significance: Enhancers confer cell-type specificity and temporal control over gene expression,
which is essential during development and differentiation.

Silencers

● Definition: DNA elements that repress gene transcription.

● Mechanism: Bind repressor proteins that inhibit the assembly or activity of the transcriptional
machinery.
● Outcome: Lead to downregulation or complete silencing of gene expression, either by preventing
activator binding, blocking enhancer function, or recruiting chromatin-condensing factors such as
histone deacetylases.

Insulators

● Definition: DNA sequences that function as genomic boundaries.

● Types:
○ Enhancer-blocking insulators: Prevent enhancers from inappropriately activating nearby
unrelated promoters.
○ Barrier insulators: Protect genes from the spread of repressive heterochromatin.
● Protein Association: Frequently bind to the CCCTC-binding factor (CTCF), a zinc-finger protein that
mediates chromatin looping and insulator activity.
 ● Role: Ensure specific and context-dependent gene regulation by maintaining the integrity of
regulatory domains.
3. Explain the role of histones in chromatin organization, including the impact of histone modifications
on gene expression.

Histones are small, positively charged proteins that play a fundamental role in the compaction, organization,
and regulation of eukaryotic DNA within the nucleus. They are essential structural components of chromatin,
which is the complex of DNA and associated proteins that forms chromosomes.

Core Structure of Chromatin

● Nucleosome: The basic unit of chromatin, consisting of 147 base pairs of DNA wrapped around an
octamer of histones—two copies each of histones H2A, H2B, H3, and H4.
● Linker DNA: Segments of DNA between nucleosomes are associated with histone H1, which helps
compact chromatin into higher-order structures.
● This packaging allows approximately 2 meters of DNA to fit within the 5–10 μm nucleus and also
plays a dynamic role in the regulation of gene accessibility and transcription.

Impact of Histone Modifications on Gene Expression

Histone proteins possess amino-terminal tails that extend outward from the nucleosome core and are subject
to various post-translational modifications (PTMs). These modifications do not change the DNA sequence
but can profoundly influence chromatin structure and gene expression, making them a central component
of the epigenetic code.

Key Histone Modifications

1. Acetylation
○ Catalyzed by: Histone acetyltransferases (HATs)
○ Target residues: Lysine residues on histone tails (e.g., H3K9, H3K27)
○ Effect: Neutralizes positive charges on histones, decreasing their affinity for negatively charged
DNA, resulting in a more relaxed chromatin structure (euchromatin).
○ Consequence: Facilitates gene transcription by allowing transcription factor access.
○ Reversed by: Histone deacetylases (HDACs), which promote chromatin condensation and
gene repression.

2. Methylation
○ Catalyzed by: Histone methyltransferases
○ Target residues: Primarily lysine and arginine residues (e.g., H3K4, H3K9, H3K27)
○ Effect: Can lead to either activation or repression of gene expression, depending on the site
and degree of methylation:
■ H3K4me3: Associated with active transcription.
■ H3K9me3 or H3K27me3: Associated with transcriptional silencing (heterochromatin
formation).
○ Reversed by: Histone demethylases.

3. Phosphorylation
○ Typically occurs on serine and threonine residues.
○ Involved in chromatin remodeling during processes such as DNA repair and mitosis, as well as
in regulating gene activity.

4. Ubiquitination and Sumoylation

○ Affect chromatin dynamics and serve as signals for DNA repair, transcriptional regulation, or
protein degradation.

Chromatin States and Gene Accessibility

Histone modifications determine the functional state of chromatin:

● Euchromatin
○ Loosely packed chromatin associated with active gene transcription.
○ Enriched with activating marks like H3K4me3 and histone acetylation.

● Heterochromatin
○ Densely packed chromatin typically associated with gene silencing.
○ Enriched with repressive marks such as H3K9me3, H3K27me3, and hypoacetylated histones.

These modifications are interpreted by “reader” proteins that recognize specific marks and recruit chromatin-
remodeling complexes, transcriptional activators or repressors, or DNA repair machinery.

4. Define micro-RNAs (miRNAs) and long noncoding RNAs (lncRNAs) and their respective roles in
gene silencing, chromatin remodeling, and post-transcriptional regulation.

Micro-RNAs (miRNAs)

● Definition: miRNAs are small, approximately 22-nucleotide-long, single-stranded noncoding RNAs that
regulate gene expression post-transcriptionally.
● Biogenesis: They are transcribed as primary transcripts (pri-miRNAs), processed into precursor
miRNAs (pre-miRNAs) in the nucleus, and further cleaved in the cytoplasm to generate mature
miRNAs.
● Function: miRNAs bind to complementary sequences on target messenger RNAs (mRNAs), primarily
in the 3′ untranslated region (3′ UTR).
● Role in Gene Silencing:
○ miRNAs inhibit gene expression by promoting mRNA degradation or repressing translation.
○ This mechanism allows fine-tuning of protein synthesis and is crucial in development,
differentiation, and cellular homeostasis.
● Impact on Disease: Dysregulation of miRNAs is implicated in various diseases, including cancer,
where they may function as oncogenes or tumor suppressors.

Long Noncoding RNAs (lncRNAs)

● Definition: lncRNAs are transcripts longer than 200 nucleotides that do not encode proteins.
● Localization: They can function in the nucleus or cytoplasm depending on their roles.
● Functions:
○ Chromatin Remodeling: lncRNAs can recruit chromatin-modifying complexes to specific
genomic loci, altering histone modifications and DNA methylation patterns to regulate gene
expression epigenetically.
○ Transcriptional Regulation: lncRNAs can act as scaffolds or guides for transcription factors
and chromatin remodelers, influencing the activation or repression of gene transcription.
○ Post-transcriptional Regulation: lncRNAs may interact with mRNAs or miRNAs, affecting
mRNA stability, splicing, or translation.
● Role in Cellular Processes: lncRNAs are involved in processes such as X-chromosome inactivation,
imprinting, and the response to stress.
● Clinical Relevance: Alterations in lncRNA expression have been linked to developmental disorders
and cancer.
 5. Discuss the principles and therapeutic applications of gene editing technologies, particularly
CRISPR-Cas9.

Gene editing refers to a group of molecular techniques designed to precisely modify DNA sequences within
the genome. These tools enable insertion, deletion, or replacement of specific DNA segments to correct
mutations or alter gene function.

CRISPR-Cas9 System

● Origin: Derived from a bacterial adaptive immune system that targets and cleaves foreign DNA.
● Components:
1. Cas9 nuclease: An enzyme that introduces double-strand breaks (DSBs) at specific DNA sites.
2. Guide RNA (gRNA): A synthetic RNA molecule that directs Cas9 to the complementary target
DNA sequence via base pairing.
● Mechanism:
1. The gRNA-Cas9 complex binds to the target DNA sequence adjacent to a protospacer adjacent
motif (PAM).
2. Cas9 introduces a DSB at the targeted location.
3. The cell repairs the break via:
■ Non-homologous end joining (NHEJ): An error-prone repair pathway that can
introduce insertions or deletions (indels), often disrupting gene function.
■ Homology-directed repair (HDR): A precise repair mechanism using a provided DNA
template to correct or insert specific sequences.

Gene editing technologies, particularly CRISPR-Cas9, offer promising strategies for treating genetic diseases
by correcting pathogenic mutations or modulating gene expression.

● Monogenic Disorders: Correction of mutations in diseases such as sickle cell anemia, cystic fibrosis,
and Duchenne muscular dystrophy.
● Cancer: Engineering immune cells (e.g., CAR-T cells) with gene editing to enhance targeting and killing
of tumor cells.
● Infectious Diseases: Potential to target viral genomes integrated into host DNA (e.g., HIV).
● Research and Diagnostics: Creating disease models for study and drug screening; improving genetic
testing accuracy.

Challenges and Considerations

● Off-target Effects: Unintended cleavage at similar DNA sequences, risking genomic instability.
● Delivery: Efficient and safe methods to deliver CRISPR components into target cells or tissues.
● Ethical Issues: Particularly regarding germline editing and long-term consequences.

II. Basic Cell Structure and Housekeeping Functions

6. Outline the protective and functional roles of the plasma membrane, including maintenance of
homeostasis, signal transduction, and selective permeability.

Protective and Functional Roles of the Plasma Membrane

1. Maintenance of Homeostasis

● The plasma membrane acts as a barrier separating the intracellular environment from the extracellular
space.
● It regulates the composition of the cytoplasm by controlling the influx and efflux of ions, nutrients,
and waste products.
● Maintains electrochemical gradients essential for cellular processes such as nutrient uptake, waste
removal, and electrical excitability.
 ● Supports cell volume regulation by controlling osmotic balance through selective transport
mechanisms.

2. Selective Permeability

● The plasma membrane is selectively permeable, allowing only certain molecules to cross.
● Small, nonpolar molecules (e.g., O₂, CO₂) diffuse freely.
● Ions and polar molecules require specific transport proteins:
○ Channels: Facilitate passive transport of ions or water.
○ Carriers/Transporters: Mediate facilitated diffusion or active transport using energy.
● This selective permeability enables the cell to control its internal environment precisely.

3. Signal Transduction

● The plasma membrane contains receptors that detect extracellular signals such as hormones,
neurotransmitters, and growth factors.
● Binding of ligands to receptors triggers intracellular signaling cascades that modulate gene
expression, metabolism, cell growth, and apoptosis.
● Receptors include G protein-coupled receptors, receptor tyrosine kinases, and ion channel-linked
receptors.
● This function allows cells to respond and adapt to changes in their environment.

4. Other Functional Roles

● Cell adhesion: Membrane proteins mediate attachment to the extracellular matrix and neighboring
cells, important for tissue structure.
● Endocytosis and exocytosis: The membrane facilitates uptake of substances (endocytosis) and
secretion of molecules (exocytosis).
● Signal amplification and integration: Membrane domains organize signaling molecules to ensure
efficient responses.

7. Classify and describe the major types of membrane transport, including passive diffusion,
facilitated diffusion, active transport, and vesicular transport.

1. Passive Diffusion

● Definition: Movement of molecules directly across the lipid bilayer down their concentration gradient
without the use of energy.
● Characteristics:
○ Occurs with small, nonpolar, and lipid-soluble molecules (e.g., oxygen, carbon dioxide, steroid
hormones).
○ Does not require membrane proteins.
● Driving Force: Concentration gradient (high to low).

2. Facilitated Diffusion

● Definition: Passive transport of molecules across the membrane via specific transmembrane proteins.
● Types of Proteins:
○ Channel proteins: Form pores that allow selective passage of ions or water.
○ Carrier proteins: Bind specific molecules and undergo conformational changes to transport
them.
● Characteristics:
○ Moves substances down their concentration gradient.
○ Does not require cellular energy (ATP).
○ Examples: Glucose transport via GLUT transporters; ion movement through ion channels.
3. Active Transport

● Definition: Movement of molecules against their concentration gradient, requiring energy input.
● Energy Source: Usually ATP hydrolysis or coupled transport.
● Types:
○ Primary active transport: Direct use of ATP to transport molecules.
■ Example: Na⁺/K⁺-ATPase pump actively pumps Na⁺ out and K⁺ into the cell.
○ Secondary active transport (cotransport): Uses the energy stored in ion gradients
established by primary active transport.
■ Symporters: Move two molecules in the same direction.
■ Antiporters: Move two molecules in opposite directions.
■ Example: Na⁺-glucose cotransporter in the intestine.

4. Vesicular Transport

● Definition: Transport of large molecules or particles via membrane-bound vesicles.

● Energy Requirement: Requires ATP for vesicle formation and trafficking
● Types:
○ Endocytosis: Uptake of extracellular material by invagination of the plasma membrane.
■ Phagocytosis: Engulfment of large particles or cells.
■ Pinocytosis: Nonspecific uptake of extracellular fluid.
■ Receptor-mediated endocytosis: Selective uptake of molecules via receptor binding.
■ Exocytosis: Fusion of vesicles with the plasma membrane to secrete substances.

8. Describe the structural components and functional importance of the cytoskeleton, including
microtubules, actin filaments, and intermediate filaments.

The cytoskeleton is a dynamic network of protein filaments that provides structural support, maintains cell
shape, enables intracellular transport, and facilitates cell movement and division. It consists of three major
types of filaments: microtubules, actin filaments (microfilaments), and intermediate filaments.

1. Microtubules

● Structure:
○ Composed of polymerized α- and β-tubulin dimers forming hollow cylindrical tubes about 25 nm
in diameter.
○ Polar structures with a plus (+) end (growing) and a minus (−) end (anchored at
microtubule-organizing centers such as the centrosome).
● Functions:
○ Serve as tracks for intracellular transport of organelles and vesicles via motor proteins
(kinesin and dynein).
○ Form the mitotic spindle during cell division, essential for chromosome segregation.
○ Provide structural support for cilia and flagella enabling cell motility.
○ Participate in maintaining cell shape and polarity.

2. Actin Filaments (Microfilaments)

● Structure:
○ Composed of polymerized globular actin (G-actin) monomers forming thin, flexible filaments
about 7 nm in diameter.
○ Polar with a fast-growing plus (+) end and a slower minus (−) end.
● Functions:
○ Maintain and modify cell shape by forming a dense network beneath the plasma membrane.
○ Facilitate cell movement via lamellipodia and filopodia during processes like migration.
○ Support cytokinesis (division of the cytoplasm) during mitosis.
 ○ Participate in endocytosis and exocytosis.
○ In muscle cells, interact with myosin for contraction.

3. Intermediate Filaments

● Structure:
○ Composed of various tissue-specific proteins (e.g., keratins in epithelial cells, vimentin in
mesenchymal cells, neurofilaments in neurons).
○ Filaments about 10 nm in diameter, more stable and less dynamic than microtubules or actin
filaments.
● Functions:
○ Provide mechanical strength to cells and tissues, resisting shear stress.
○ Anchor the nucleus and other organelles within the cytoplasm.
○ Contribute to the formation of desmosomes and hemidesmosomes, structures important for
cell-cell and cell-matrix adhesion.

9. Summarize the role of cell-cell junctions and adhesion molecules in maintaining tissue integrity
and intercellular communication.

Cells within tissues are connected by specialized structures called cell-cell junctions, which, together with
adhesion molecules, maintain tissue architecture, provide mechanical stability, and enable communication
between cells.

1. Types of Cell-Cell Junctions

● Tight Junctions (Zonula Occludens):

○ Seal adjacent cells near their apical surface.
○ Prevent passage of molecules between cells (paracellular barrier).
○ Maintain cell polarity by separating apical and basolateral membrane domains.

● Adherens Junctions (Zonula Adherens):

○ Connect the actin cytoskeletons of adjacent cells.
○ Involve cadherin proteins that mediate calcium-dependent cell adhesion.
○ Provide mechanical strength and contribute to tissue morphogenesis.

● Desmosomes (Macula Adherens):

○ Connect intermediate filaments between neighboring cells.
○ Provide strong adhesion to resist mechanical stress.
○ Important in tissues subjected to stretching (e.g., skin, heart).

● Gap Junctions:
○ Form channels allowing direct cytoplasmic exchange of ions and small molecules.
○ Enable electrical and metabolic coupling for intercellular communication.

2. Adhesion Molecules

● Cadherins:
○ Mediate homophilic, calcium-dependent adhesion at adherens junctions and desmosomes.
○ Important for tissue organization and signaling.

● Integrins:
○ Mediate cell-extracellular matrix adhesion
○ Involved in signaling pathways regulating cell survival, proliferation, and migration.

● Selectins and Immunoglobulin Superfamily Molecules:

 ○ Participate in transient cell-cell adhesion, especially in immune responses.

Functional Importance
● Maintain Tissue Integrity: By physically linking cells, junctions provide cohesion and mechanical
stability necessary for tissue function.
● Regulate Permeability and Polarity: Tight junctions control passage of substances, preserving
distinct membrane domains.
● Facilitate Intercellular Communication: Gap junctions enable coordinated cellular responses.
● Influence Cell Behavior: Adhesion molecules participate in signaling that affects cell growth,
differentiation, and survival.

10. Explain the biosynthetic and trafficking roles of the endoplasmic reticulum (ER) and Golgi
apparatus, and their contribution to protein folding and post-translational modification.
11. Differentiate the roles of lysosomes and proteasomes in intracellular waste disposal and protein
turnover.
III. Cellular Metabolism and Mitochondrial Function

12. Describe the structure and function of mitochondria in ATP generation, apoptosis, and reactive
oxygen species (ROS) metabolism.

13. Explain the significance of mitochondrial DNA and its unique inheritance, particularly in relation to
inherited metabolic disorders.

IV. Cellular Activation and Signaling

14. Define the major types of cellular receptors and their ligand interactions, including receptor
tyrosine kinases and G-protein–coupled receptors.

15. Outline the steps in a typical signal transduction cascade, including second messengers and
phosphorylation events.

16. Describe the role of modular signaling proteins, hubs, and nodes in integrating multiple signaling
pathways.

17. List key families of transcription factors and explain their downstream effects on gene expression
and cell fate decisions.

18. Explain how growth factors and their receptors regulate cell survival, proliferation, and differentiation.

V. Extracellular Matrix and Cell-Matrix Interactions

19. Identify the major components of the extracellular matrix (ECM)—collagens, elastin,
proteoglycans, and adhesive glycoproteins.

20. Explain the role of the ECM in tissue structure, signaling, and repair, and how integrins mediate
cell-matrix communication.

21. Distinguish between the interstitial matrix and basement membrane in terms of structure, location,
and function.

VI. Cell Proliferation and Stem Cells

22. Outline the phases of the cell cycle and the key checkpoints regulating cell division.
23. Describe the roles of cyclins, cyclin-dependent kinases (CDKs), and tumor suppressor proteins
(e.g., p53, RB) in regulating proliferation.

24. Define stem cells and distinguish between embryonic and adult (somatic) stem cells, including
their potency and self-renewal capabilities.

25. Explain the concept of asymmetric division and niche signaling in stem cell maintenance.

26. Discuss the principles and challenges of regenerative medicine, including the use of induced
pluripotent stem cells (iPSCs).