Exam 1: Organization of Chromatin - MOLECULAR Biology PDF

**Exam 1** **1. Organization of Chromatin -- Nucleosomes -- Histones** - **Chromatin:** - Complex of DNA and proteins (mainly histones) that organizes and compacts DNA in the nucleus. - **Nucleosome:** - Fundamental unit of chromatin, consisting of **147 base pairs of...

**Exam 1** **1. Organization of Chromatin -- Nucleosomes -- Histones** - **Chromatin:** - Complex of DNA and proteins (mainly histones) that organizes and compacts DNA in the nucleus. - **Nucleosome:** - Fundamental unit of chromatin, consisting of **147 base pairs of DNA** wrapped around a core of **8 histone proteins** (octamer: 2 each of H2A, H2B, H3, H4). - **H1 histone** links nucleosomes together, forming higher-order structures. - **Levels of Chromatin Organization:** - - - **2. Types of Chromatin** - **Euchromatin:** - Loosely packed, transcriptionally active regions of chromatin. - **Heterochromatin:** - Densely packed, transcriptionally inactive regions. - Subtypes: - **Constitutive Heterochromatin:** Always inactive (e.g., centromeres, telomeres). - **Facultative Heterochromatin:** Can switch between active and inactive states (e.g., X-chromosome inactivation). **3. Gene and Sequence Copy Number/Complexity in Eukaryotic Genomes** - **Unique Sequences:** Single-copy genes, coding for proteins. - **Repetitive Sequences:** - **Moderately repetitive:** Includes rDNA, tRNA, and histone genes. - **Highly repetitive:** Found in centromeres and telomeres (e.g., satellite DNA). - Complexity arises from both coding and non-coding regions. **4. Nature of the Nucleolus** - **Definition:** - A subnuclear structure where **ribosomal RNA (rRNA)** is synthesized and ribosome assembly begins. - **rDNA Genes Location:** - Found in tandem arrays on acrocentric chromosomes. - **Function:** - Transcription of rRNA by **RNA polymerase I** and assembly of ribosomal subunits. **5. Consequences of Genetic Rearrangements Involving Different Types of Chromatin** - Rearrangements in heterochromatin (e.g., translocations, inversions): - Often **silent** due to its inactive state. - Rearrangements in euchromatin: - Can **disrupt genes** or regulatory elements, leading to diseases like cancer. **6. Posttranslational Modifications and Complexes Affecting Chromatin** - **Histone Modifications:** 1. **Acetylation (by HATs):** Activates transcription by loosening chromatin. 2. **Methylation (by HMTs):** Can activate or repress transcription depending on the site. 3. **Phosphorylation:** Associated with chromatin condensation during mitosis. 4. **Ubiquitination:** Marks histones for remodeling or degradation. - **Chromatin Remodeling Complexes:** 1. ATP-dependent complexes (e.g., SWI/SNF) reposition or eject nucleosomes to allow access to DNA. **7. Functional Parts of Chromosomes** 1. **Telomeres:** - Repetitive sequences (e.g., TTAGGG in humans) at chromosome ends. - Prevent degradation and ensure complete replication. - Maintained by **telomerase**. 2. **Centromeres:** - Region where kinetochores attach, enabling proper chromosome segregation during mitosis. - Composed of heterochromatin and satellite DNA. 3. **Origins of Replication:** - Specific sequences where DNA replication begins. - Eukaryotic chromosomes have **multiple origins**. **8. DNA Replication and Repair -- Nature of Replication Process** - **Replication Process:** - Semi-conservative: Each new DNA molecule consists of one parental and one new strand. - Bi-directional: Proceeds from multiple origins of replication. - Enzymes involved: 1. **Helicase:** Unwinds DNA. 2. **Primase:** Lays down RNA primers. 3. **DNA Polymerase:** Adds nucleotides to the growing strand. 4. **Ligase:** Seals nicks in the DNA backbone. - **Continuous vs. Lagging Replication:** - **Leading Strand:** Synthesized continuously in the 5\'→3\' direction. - **Lagging Strand:** Synthesized discontinuously as **Okazaki fragments**, later joined by ligase. **9. Basic Relationship of DNA Replication to PCR** - PCR (Polymerase Chain Reaction) mimics the natural DNA replication process but in vitro: 1. **Denaturation:** DNA strands are separated by heat. 2. **Annealing:** Primers bind to complementary sequences. 3. **Extension:** DNA polymerase (e.g., Taq polymerase) synthesizes new strands. - Relies on the same principle of base-pairing and strand elongation. **Exam 2** **Types of Chromatin in Eukaryotic Chromosomes** In eukaryotes, chromatin is a complex of DNA and proteins, primarily histones, and exists in two main forms: - **Euchromatin**: This is the less condensed form of chromatin and is **transcriptionally active**. Euchromatin is typically found in regions of the genome where genes are actively being expressed. The open configuration allows transcription factors and other regulatory proteins to access the DNA. - Used for transcription in the DNA - **Heterochromatin**: This is more condensed and **transcriptionally inactive.** Heterochromatin is found in regions of the genome that are not being actively transcribed, such as centromeres and telomeres. It is further divided into: - **Constitutive heterochromatin**: Permanently inactivated and always compacted. - **Facultative heterochromatin**: Can switch between inactive and active states depending on cellular conditions. **2. Position Effect Variegation (PEV)** Position effect variegation refers to the phenomenon where the expression of a gene is affected by its location within the genome. If a gene that is normally in euchromatin is relocated close to heterochromatin (due to a chromosomal rearrangement, for example), it can become transcriptionally silenced due to the spreading of the heterochromatin. This phenomenon shows that gene expression can be influenced by chromatin structure and the gene's relative position in the genome. **3. Posttranslational Modifications and Complexes that Affect Chromatin Structure** Chromatin structure and function can be heavily influenced by **posttranslational modifications (PTMs)** on histone proteins. Some of these modifications include: - **Acetylation**: Addition of an acetyl group to lysine residues on histones (often associated with gene activation). - **Methylation**: Addition of methyl groups to lysines or arginines, which can either activate or repress transcription depending on the specific amino acid and context. - **Phosphorylation**: Addition of phosphate groups can change histone structure and interactions, often linked to DNA damage repair. - **Ubiquitination**: Addition of ubiquitin to histones can mark them for degradation or affect transcriptional activity. Several protein complexes also influence chromatin structure, such as: - **SWI/SNF complex**: A chromatin remodeling complex that uses ATP to reposition nucleosomes, making DNA more or less accessible for transcription. - **Polycomb and Trithorax groups**: Polycomb repressive complexes (PRC) promote chromatin compaction and gene silencing, while Trithorax complexes antagonize this by promoting active chromatin states. **4. Definition of an Operon** An **operon** is a functioning unit of genomic DNA that contains a cluster of genes under the control of a single promoter. Operons are common in prokaryotes and allow for coordinated regulation of gene expression. Genes within an operon are transcribed together as a single mRNA strand and often participate in related biological processes (e.g., the lac operon for lactose metabolism in *E. coli*). **5. Difference Between Parallel and Serial Analyses of Genes** - **Parallel analysis**: Involves the simultaneous examination of multiple genes. Techniques like **DNA microarrays**and **RNAseq** fall under this category as they allow for the study of thousands of genes at once. This method is useful for gaining a broad view of gene expression profiles. - **Serial analysis**: Involves examining one gene or a few genes at a time. Traditional methods like **Northern blotting**or **RT-PCR** would be examples of serial analysis techniques. These are useful when focusing on the expression of specific genes rather than genome-wide profiles. **6. Analyzing Differential Gene Expression Using DNA Microarrays or RNAseq** - **DNA microarrays**: These are glass slides or silicon chips onto which thousands of DNA probes (representing different genes) are fixed. When a sample's mRNA is extracted, converted to cDNA, and fluorescently labeled, it can be hybridized to the probes on the array. Differences in gene expression between samples (e.g., healthy vs. diseased tissue) can be visualized as differences in fluorescence intensity at specific spots on the array. - **RNAseq**: This is a high-throughput sequencing method used to quantify gene expression. mRNA is isolated from a sample, converted to cDNA, and then sequenced. RNAseq provides a more detailed and quantitative view of gene expression than microarrays, as it can measure not only gene abundance but also alternative splicing events and novel transcripts. **7. Types of RNAs Found in Eukaryotes** - **mRNA (messenger RNA)**: Carries the genetic code from DNA to the ribosome for protein synthesis. - **tRNA (transfer RNA)**: Brings the correct amino acid to the ribosome during translation. - **rRNA (ribosomal RNA)**: Forms part of the ribosome and plays a structural and catalytic role in protein synthesis. - **snRNA (small nuclear RNA)**: Involved in splicing of pre-mRNA in the nucleus. - **snoRNA (small nucleolar RNA)**: Plays a role in the chemical modification of rRNA. - **miRNA (microRNA)**: Involved in posttranscriptional regulation by binding to target mRNAs and inhibiting their translation or leading to their degradation. - **siRNA (small interfering RNA)**: Similar to miRNA, but often exogenously introduced, and it leads to the degradation of target mRNA. **8. Role of TFID and TFIH in RNA Polymerase II Transcription Initiation** - **TFID**: This is a complex that contains the TATA-binding protein (TBP) and other associated factors (TAFs). It binds to the promoter region of a gene (specifically the TATA box) and recruits RNA polymerase II and other general transcription factors to the promoter, forming the transcription initiation complex. - **TFIH**: Has both helicase and kinase activity. Its helicase function unwinds the DNA at the transcription start site, allowing RNA polymerase II to access the template strand. Its kinase function phosphorylates the C-terminal domain of RNA polymerase II, which is necessary for transcription to proceed. **9. How Activators and Repressors Regulate Gene Expression** - **Activators**: These are proteins that bind to specific DNA sequences called enhancers or promoter regions and help recruit transcription machinery (including RNA polymerase II) to start transcription. Activators often interact with coactivators and mediator complexes, which help facilitate chromatin remodeling and transcription initiation. - **Repressors**: Bind to specific DNA sequences (like silencers) or interact with co-repressors to block the recruitment of transcriptional machinery or condense chromatin, preventing transcription. Some repressors also compete with activators by blocking their binding sites. **10. Purpose of Having a Variety of Activators and Repressors and Multiple Points of Regulation** The use of different activators, repressors, and regulatory mechanisms allows cells to finely tune gene expression in response to various internal and external signals. This flexibility ensures that genes are expressed at the right time, place, and level to meet the organism's needs. Additionally, it allows for tissue-specific gene expression and the ability to respond rapidly to changing conditions. **11. Introns and Alternative Splicing** - **Introns**: These are non-coding sequences within genes that are removed during mRNA processing in the nucleus. The removal of introns is performed by the **spliceosome**. - **Alternative splicing**: Refers to the process where different combinations of exons are joined together, resulting in the production of different protein isoforms from a single gene. This increases the diversity of proteins that a single gene can produce and is critical for tissue-specific functions. **12. Posttranscriptional Gene Expression Control Mechanisms** Posttranscriptional regulation includes: - **RNA splicing**: The removal of introns and joining of exons. - **RNA transport**: The export of mRNA from the nucleus to the cytoplasm. - **RNA stability**: The half-life of mRNA can be regulated, affecting how long it remains available for translation. - **RNA interference**: Small RNAs like miRNAs and siRNAs can degrade mRNA or inhibit translation. **13. Steps of Translation in Eukaryotes** - **Initiation**: The small ribosomal subunit binds to the mRNA, with the help of initiation factors. The initiator tRNA, carrying methionine, binds to the start codon (AUG). The large ribosomal subunit then joins. - **Elongation**: The ribosome moves along the mRNA, and new tRNAs bring amino acids to the ribosome. The ribosome has three sites: - **A site**: Aminoacyl-tRNA site where incoming tRNAs bind. - **P site**: Peptidyl-tRNA site where the growing peptide chain is held. - **E site**: Exit site where empty tRNAs leave the ribosome. - **Termination**: When a stop codon is encountered, release factors bind, and the polypeptide chain is released. **14. Nature of the Genetic Code** The genetic code consists of sequences of three nucleotides (codons), each of which codes for one amino acid. There are **64 possible codons** for the 20 amino acids, with **start codons** (AUG for methionine) and **stop codons** (UAA, UAG, UGA) signaling the initiation and **LECTURE 5 --- Tools to study genes and gene products (Chapter 8)** **CRISPR-Cas9: Target and Mechanism** CRISPR-Cas9 is a tool used to edit **DNA**. It originates from a bacterial immune system where it protects against viruses by cutting foreign DNA. In gene editing, CRISPR-Cas9 can be programmed to target specific DNA sequences through the use of a **guide RNA (gRNA)**. This RNA molecule is designed to be complementary to the target DNA sequence, ensuring specificity. Once the Cas9 protein is directed to the specific location, it introduces a double-stranded break in the DNA. The cell then repairs this break through one of two pathways: - **Non-homologous end joining (NHEJ)**: Often results in insertions or deletions (indels), which can disrupt gene function. - **Homology-directed repair (HDR)**: Allows the insertion of a specific DNA sequence if a donor template is provided, enabling precise gene modifications. **2. Southern, Northern, and Western Blots** Each blotting technique is used to analyze different types of macromolecules: - **Southern Blot**: Detects **DNA**. This method involves digesting DNA with restriction enzymes, separating the fragments via gel electrophoresis, transferring the DNA to a membrane, and probing with a labeled DNA or RNA sequence that binds to a specific DNA fragment. - **Northern Blot**: Detects **RNA**. RNA molecules are separated by size using gel electrophoresis, transferred to a membrane, and probed with a labeled complementary RNA or DNA strand. This technique is commonly used to study gene expression by detecting the presence and size of specific mRNA transcripts. - **Western Blot**: Detects **proteins**. Proteins are separated based on size by gel electrophoresis, transferred to a membrane, and probed using specific antibodies that bind to the target protein. A secondary antibody with an enzyme or fluorescent label is then used for detection. **3. GFP, FRAP, FRET, and Antibodies in Microscopy** - **GFP (Green Fluorescent Protein)**: GFP is a protein that fluoresces green when exposed to UV or blue light. It is often used as a **tag** to visualize proteins in living cells. By fusing GFP to proteins of interest, researchers can track protein localization and dynamics within cells. - **FRAP (Fluorescence Recovery After Photobleaching)**: FRAP is used to study the **mobility** of fluorescently labeled molecules within cells. A specific region of the cell is photobleached with intense light, and the recovery of fluorescence in the area is monitored over time as unbleached molecules move into the area, indicating molecular dynamics. - **FRET (Fluorescence Resonance Energy Transfer)**: FRET is used to study **interactions between proteins or other molecules**. It works when two fluorescent proteins are in close proximity; energy is transferred from one (the donor) to the other (the acceptor), producing a detectable signal. This technique is valuable for studying protein-protein interactions and conformational changes. - **Antibodies in Electron Microscopy and Fluorescence Microscopy**: Antibodies can be used in both techniques to **specifically bind to proteins of interest**. In fluorescence microscopy, antibodies are labeled with fluorescent dyes, enabling the visualization of specific proteins within cells. In electron microscopy (EM), antibodies are often tagged with gold particles, which appear as electron-dense spots, allowing precise localization of proteins at the ultrastructural level. **4. Obtaining Images for Electron Microscopy** Electron microscopy (EM) uses electrons rather than light to generate high-resolution images. There are two main types of EM: - **Transmission Electron Microscopy (TEM)**: This method passes electrons through thin sections of a sample. Electrons interact with the sample and form an image based on the electron density of the material. TEM provides detailed information about the internal structure of cells and organelles. - **Scanning Electron Microscopy (SEM)**: In SEM, a beam of electrons scans the surface of a specimen. Electrons that are scattered from the surface create a 3D image, showing the surface topography of the sample. Samples for EM need special preparation, including dehydration, embedding, sectioning (for TEM), and coating with heavy metals (for SEM) to increase electron contrast. **5. Chromatography: Types and Uses in Protein Separation** - **Ion-exchange Chromatography**: This technique separates proteins based on their **charge**. Proteins are passed through a column containing charged beads (either positive or negative). Proteins with an opposite charge to the beads bind, while others flow through. Bound proteins are then eluted by changing the ionic strength of the buffer. - **Size-exclusion Chromatography**: Also known as **gel filtration**, this method separates proteins based on their **size**. The column contains porous beads; small molecules enter the pores and move through the column more slowly, while larger molecules are excluded from the pores and elute faster. - **Affinity Chromatography**: This technique relies on **specific interactions** between a protein and a ligand (e.g., an antibody or a substrate analogue) that is immobilized on the beads. Proteins that have a high affinity for the ligand will bind, while others are washed away. The bound protein is then eluted by adding a competitive ligand or changing conditions (e.g., pH). **6. Mass Spectrometry in Protein Identification** Mass spectrometry (MS) is used to identify proteins by determining their **mass-to-charge (m/z) ratio**. The general steps in protein identification are: - **Ionization**: Proteins or peptides are ionized (often by electrospray ionization or matrix-assisted laser desorption/ionization, MALDI). - **Mass Analyzer**: The ions are passed through a mass analyzer, which separates them based on their m/z ratio. - **Detection**: The detector records the mass spectrum, which shows the abundance of ions at different m/z values. The resulting spectrum can be matched to known proteins or peptides using databases. Mass spectrometry is highly sensitive and allows for the identification of proteins, post-translational modifications, and even protein-protein interactions. **7. Nature of Different Forms of Mutations and Genetic Variation** **Point Mutations** - **Definition**: A point mutation is a change in a single nucleotide base in the DNA sequence. - **Types**: - **Silent mutation**: Does not change the amino acid sequence due to the redundancy of the genetic code (synonymous change). - **Missense mutation**: Alters one amino acid in the protein (non-synonymous change), which can affect protein function depending on the location and nature of the change. - **Nonsense mutation**: Converts a codon into a stop codon, leading to a truncated and usually nonfunctional protein. - **Consequences**: Point mutations can lead to diseases if they occur in essential genes or regulatory regions, or they can be neutral if they occur in non-coding regions or do not significantly affect protein function. **Chromosomal Rearrangements** - **Types**: - **Deletions**: Loss of a chromosome segment, which can lead to the loss of essential genes. - **Duplications**: Repetition of a chromosome segment, which can result in gene dosage effects. - **Inversions**: A segment of the chromosome is reversed. If breakpoints occur within a gene, it can disrupt gene function. - **Translocations**: Segments from two different chromosomes are exchanged, potentially leading to gene fusion or misregulation of genes. - **Consequences**: Chromosomal rearrangements can lead to developmental disorders, cancer (e.g., fusion of oncogenes), or have little effect if they do not disrupt essential genes. **Aneuploidy** - **Definition**: The presence of an abnormal number of chromosomes (more or fewer than the typical diploid number). - **Types**: - **Monosomy**: Loss of one chromosome (e.g., Turner syndrome -- 45, X). - **Trisomy**: Presence of an extra chromosome (e.g., Down syndrome -- Trisomy 21). - **Consequences**: Aneuploidy often leads to developmental abnormalities, miscarriage, or disease, as the balance of gene expression is disrupted. **Polyploidy** - **Definition**: The condition of having more than two complete sets of chromosomes. - **Types**: - **Triploidy**: Three sets of chromosomes (3n). - **Tetraploidy**: Four sets of chromosomes (4n). - **Consequences**: Polyploidy is more common in plants and can result in larger, more robust species. In animals, polyploidy is often lethal or results in sterility (e.g., triploid fish). **LECTURE 6 --- Nature of Cell Membranes, some tools to study genes and gene products (Chapter 11 and 15)** **1. Composition and Chemical Nature of Cellular Membranes** Cellular membranes are primarily composed of **lipids**, **proteins**, and **carbohydrates**. The main types of lipids found in membranes are phospholipids, glycolipids, and sterols (such as cholesterol). These components have different chemical properties: - **Phospholipids**: They are **amphipathic**, meaning they have both hydrophilic (water-attracting) and hydrophobic (water-repelling) parts. The hydrophilic **head** group is polar and faces the aqueous environment, while the hydrophobic **tail** group, consisting of fatty acid chains, is non-polar and points inward, away from water. This arrangement forms a **lipid bilayer**. - **Glycolipids**: These lipids have a **polar carbohydrate** group attached to a lipid tail. Like phospholipids, they are amphipathic and contribute to the cell membrane\'s asymmetry. - **Sterols (e.g., Cholesterol)**: Cholesterol has a **polar hydroxyl group** that interacts with the hydrophilic heads of phospholipids, while its **non-polar steroid rings and hydrocarbon tail** interact with the fatty acid tails. Cholesterol modulates membrane fluidity. - **Proteins**: Membrane proteins can be either **integral** (embedded within the membrane) or **peripheral** (loosely associated with the membrane surface). Integral membrane proteins are amphipathic, with hydrophobic regions interacting with the lipid tails and hydrophilic regions exposed to the aqueous environment. Peripheral proteins are polar and interact with the membrane\'s surface or other proteins. **2. Role of Hydrocarbon Tail Length and Unsaturation in Membrane Fluidity** The **fluidity** of a membrane is influenced by the properties of the fatty acid tails of phospholipids: - **Tail Length**: Shorter hydrocarbon tails reduce the van der Waals interactions between adjacent phospholipids, leading to increased membrane fluidity. Longer tails have stronger interactions, making the membrane more rigid. - **Saturation**: **Saturated** fatty acids have no double bonds, allowing them to pack tightly together, reducing membrane fluidity. **Unsaturated** fatty acids contain one or more **double bonds**, which introduce kinks in the hydrocarbon chain, preventing tight packing. This increases fluidity, making the membrane more flexible and dynamic. **3. Effects of Cholesterol on Membrane Fluidity** Cholesterol plays a crucial role in regulating membrane fluidity and stability: - **At high temperatures**, cholesterol decreases membrane fluidity by stabilizing interactions between phospholipids, preventing the membrane from becoming too fluid and permeable. - **At low temperatures**, cholesterol prevents the membrane from becoming too rigid by disrupting the close packing of phospholipids, thus maintaining fluidity. Overall, cholesterol acts as a buffer, keeping membrane fluidity within an optimal range across various temperatures. **4. Movements of Lipids and Proteins in Membranes** Lipids and proteins can move within the membrane, but their movement is constrained by the membrane's bilayer structure: - **Lateral Diffusion**: Lipids and proteins can move laterally within the same leaflet of the bilayer. This movement is relatively fast and occurs freely within the plane of the membrane. - **Flip-flop**: The movement of lipids from one leaflet of the bilayer to the other is rare because it requires the hydrophilic head group to pass through the hydrophobic core of the membrane. Special enzymes called **flippases**, **floppases**, and **scramblases** facilitate this process (more below). - **Protein Movement**: Membrane proteins also move laterally, but their movement can be restricted by **anchoring** to the cytoskeleton or extracellular matrix, or by **protein-protein interactions**. **5. Proteins that Facilitate Phospholipid Movement** The movement of phospholipids between the two leaflets of the membrane is mediated by specific proteins: - **Flippases**: These enzymes facilitate the movement of phospholipids from the outer leaflet to the inner leaflet of the bilayer. They use ATP to catalyze this energy-dependent process. - **Floppases**: These enzymes move phospholipids from the inner leaflet to the outer leaflet, also in an ATP-dependent manner. - **Scramblases**: These proteins facilitate the bidirectional movement of phospholipids between the leaflets in a non-specific and energy-independent manner. Scramblases are typically activated during apoptosis or other forms of cellular stress, disrupting membrane asymmetry. **6. Cellular Location of Phospholipid Synthesis** Phospholipids are synthesized in the **smooth endoplasmic reticulum (ER)**. The newly synthesized phospholipids are inserted into the cytoplasmic leaflet of the ER membrane. To maintain membrane symmetry, **flippases** transport some of these phospholipids to the opposite leaflet. From the ER, vesicles containing lipids and proteins are sent to other parts of the cell, including the Golgi apparatus, plasma membrane, and various organelles. **7. Sidedness of Membranes in Vesicle Budding and Docking** Membranes have **asymmetry**, meaning that the two leaflets of the bilayer have different lipid and protein compositions. This asymmetry is maintained during vesicle trafficking: - When a vesicle buds off from an organelle (like the Golgi apparatus), the leaflet that was **facing the cytoplasm** in the donor membrane continues to face the cytoplasm in the vesicle. - Upon **docking and fusion** with the plasma membrane, the vesicle\'s inner leaflet (which faced the vesicle lumen) becomes part of the outer leaflet of the plasma membrane, while the outer leaflet remains facing the cytoplasm. This preserves the asymmetric distribution of lipids and proteins. **8. Assessing Movement or Anchoring of Membrane Proteins** The movement and diffusion rates of membrane proteins can be studied using various techniques: - **Fluorescence Recovery After Photobleaching (FRAP)**: In this method, membrane proteins tagged with fluorescent markers are bleached in a specific region of the membrane using intense light. The recovery of fluorescence in the bleached area, due to the movement of unbleached proteins, provides information about the **diffusion rates** and mobility of the proteins. - **Single-Particle Tracking (SPT)**: In this technique, individual membrane proteins are tagged with fluorescent or gold markers, and their movement is tracked over time using high-resolution microscopy. SPT can reveal if the proteins move freely, are anchored, or are confined within membrane domains. **9. Integral vs. Peripheral Membrane Proteins** Membrane proteins can be classified based on how they associate with the membrane: - **Integral Membrane Proteins**: These proteins are embedded within the lipid bilayer, often spanning it completely. They can be **single-pass** or **multi-pass** transmembrane proteins. Because of their hydrophobic interactions with the lipid tails, they are tightly bound and require **detergents** to solubilize and extract them from the membrane. - **Peripheral Membrane Proteins**: These proteins are not embedded in the lipid bilayer but are instead loosely associated with the membrane surface, often through interactions with integral membrane proteins or polar head groups of lipids. Peripheral proteins can be released from the membrane by treatment with **high salt** concentrations or **pH changes**, which disrupt the electrostatic or hydrogen bond interactions. In summary, the distinction between integral and peripheral membrane proteins can be experimentally determined by their solubility in detergents versus salt solutions. Integral proteins require harsher treatment with detergents, while peripheral proteins can be extracted with milder treatments such as salts **LECTURE 7 --- Protein trafficking and cellular compartments (Chapter 15)** **1. Nature and Location of Signals for Nuclear Import** Proteins destined for the **nucleus** contain specific sequences known as **Nuclear Localization Signals (NLS)**. These signals are generally short, rich in **positively charged amino acids** like lysine and arginine, and can be located almost anywhere in the protein's sequence. The most common NLS is a **short stretch of basic residues**, but more complex, bipartite signals (two separate clusters of basic amino acids) also exist. The NLS is recognized by **importin**, a transport receptor that binds to the cargo protein in the cytoplasm and facilitates its transport through the **nuclear pore complex (NPC)** into the nucleus. Once inside, the complex dissociates, and importin is recycled back to the cytoplasm for further use. **2. Nature and Location of Signals for ER Import and ER Retention** Proteins destined for the **endoplasmic reticulum (ER)** usually contain a **signal sequence** at their **N-terminus**. This **ER signal sequence** is typically composed of about 15--30 hydrophobic amino acids. Once synthesized, this signal sequence is recognized by the **signal recognition particle (SRP)** in the cytoplasm, which halts translation and directs the ribosome to the ER membrane. Upon docking with the **SRP receptor** on the ER membrane, translation resumes, and the growing polypeptide is translocated into the ER lumen through a channel called the **translocon**. Once in the ER, the signal sequence is usually cleaved off by a **signal peptidase**. For proteins that are meant to **remain in the ER**, there is an additional signal called the **ER retention signal**. A well-known retention signal is the **KDEL** sequence (Lys-Asp-Glu-Leu), which is recognized by receptors in the Golgi apparatus, ensuring that the protein is returned to the ER if it escapes. **3. Nature and Location of Signals for Mitochondrial or Chloroplast Import** Proteins destined for the **mitochondria** or **chloroplasts** have specific **targeting signals** that guide them to these organelles. These signals are usually located at the **N-terminus** and are rich in **positively charged and hydroxylated amino acids**. - **Mitochondrial Import**: Mitochondrial proteins contain an **N-terminal presequence** that is usually amphipathic, meaning one side of the helix is hydrophobic while the other is positively charged. This signal is recognized by the **TOM (Translocase of the Outer Membrane)** complex on the mitochondrial outer membrane, which helps to translocate the protein into the intermembrane space or across into the matrix. - **Chloroplast Import**: Chloroplast proteins contain a similar **N-terminal transit peptide** that directs them to the chloroplast. The protein passes through the **TOC (Translocon of the Outer Chloroplast Membrane)** and **TIC (Translocon of the Inner Chloroplast Membrane)** complexes for import into the stroma or thylakoid membranes. **4. Folded States of Proteins During Import into the Above Compartments** During import into organelles like the ER, mitochondria, and chloroplasts, proteins are typically imported in an **unfolded state**: - **Nuclear import** allows proteins to enter the nucleus in their **fully folded state** because the nuclear pores are large enough to accommodate folded proteins. - **Mitochondrial and chloroplast import** require proteins to be kept in an unfolded state. **Chaperone proteins** such as **Hsp70** bind to the precursor proteins in the cytoplasm to prevent premature folding. Once the protein has been translocated into the matrix or stroma, additional chaperones help refold the protein into its functional state. - **ER import**: Proteins destined for the ER are often co-translationally imported, meaning they are translocated into the ER lumen as they are synthesized, remaining mostly unfolded until they are fully translocated and then folded with the help of **chaperones** in the ER lumen. **5. Nuclear Pores and Energy Requirements for Import Receptor Recycling** The **nuclear pore complex (NPC)** allows for the transport of large macromolecules into and out of the nucleus. **Nuclear import receptors** such as **importins** bind to the cargo protein with an NLS in the cytoplasm, pass through the nuclear pore, and release the cargo inside the nucleus. The process requires **energy**, which is supplied by the small GTPase **Ran**. In the nucleus, **Ran-GTP** binds to importin, causing it to release its cargo. The importin-Ran-GTP complex is then exported back to the cytoplasm, where **Ran-GTP**is hydrolyzed to **Ran-GDP**, causing importin to dissociate. Importin is then free to bind another cargo protein for import. **6. Removal or Not of Signal Sequences from Imported Proteins** - **Nuclear import**: Signal sequences such as NLS are **not removed** after transport. NLS sequences are part of the protein's permanent structure because they can be used repeatedly for nuclear import (especially during the cell cycle). - **ER import**: The N-terminal signal sequence that directs a protein to the ER is typically **cleaved off** by a **signal peptidase** once the protein has entered the ER lumen. - **Mitochondrial and chloroplast import**: The N-terminal targeting sequences are usually **removed** after import by **proteases** in the matrix or stroma, respectively. This ensures that the signal sequence doesn't interfere with the protein's function. **7. Exocytosis and Endocytosis** - **Exocytosis**: This process involves the fusion of vesicles with the **plasma membrane** to release their contents outside the cell. Exocytosis is essential for processes such as neurotransmitter release, hormone secretion, and the insertion of membrane proteins into the plasma membrane. - **Endocytosis**: The process by which the cell **engulfs external materials** by folding the plasma membrane inward to form a vesicle. This is crucial for nutrient uptake, receptor recycling, and removing membrane proteins. There are different types of endocytosis, including **phagocytosis** (cell eating), **pinocytosis** (cell drinking), and **receptor-mediated endocytosis** (specific uptake of molecules). **8. General Requirements for Vesicle Formation, Targeting, and Fusion** - **Vesicle Formation**: Requires a **coating protein** (e.g., **clathrin**, **COPI**, **COPII**) that helps deform the membrane to form a vesicle. **Dynamin** is a protein that helps pinch off the vesicle from the donor membrane. - **Vesicle Targeting**: Vesicles carry specific **Rab GTPases** on their surface, which act as molecular zip codes to ensure that the vesicle reaches the correct destination. **Tethering proteins** at the target membrane interact with Rab proteins to dock the vesicle. - **Vesicle Fusion**: **SNARE proteins** (v-SNAREs on the vesicle and t-SNAREs on the target membrane) mediate the fusion of the vesicle with the target membrane. The SNAREs wrap around each other, pulling the membranes together to facilitate fusion. **9. Unfolded Protein Response and Fates of Improperly Folded Proteins** The **Unfolded Protein Response (UPR)** is activated when misfolded proteins accumulate in the ER. It helps restore normal function by: - **Slowing down protein synthesis** to reduce the burden on the ER. - **Increasing the production of molecular chaperones** that assist in protein folding. - If the stress persists, triggering **apoptosis** to remove the affected cell. Misfolded proteins that cannot be refolded are tagged with **ubiquitin** and degraded by the **proteasome** through a process called **ER-associated degradation (ERAD)**. **10. SNAREs, Rabs, Coats -- General Roles** - **SNAREs**: These proteins mediate vesicle fusion by bringing the vesicle and target membranes together. **v-SNAREs**on the vesicle and **t-SNAREs** on the target membrane form a tight complex that facilitates membrane fusion. - **Rabs**: These small GTPases regulate vesicle **targeting** and **docking** by interacting with specific effector proteins on target membranes, ensuring that vesicles fuse with the correct compartment. - **Coats**: Proteins like **clathrin**, **COPI**, and **COPII** coat vesicles during formation, shaping the membrane and selecting the cargo for transport. **Clathrin** mediates endocytosis, while **COPI** and **COPII** are involved in trafficking between the ER and Golgi. **11. Default Location of Proteins Lacking Sorting Signals** Proteins that lack specific sorting signals are typically transported by default to the **cytoplasm**. If no signal sequence directs a protein to organelles like the ER, mitochondria, or nucleus, it remains in the cytoplasm where it was synthesized. Similarly, proteins without additional signals for vesicular trafficking will be secreted outside the cell via the constitutive secretory pathway. **EXAM 3** **LECTURE (Chapter 17 and 18) --- Cytoskeleton and Cell Division** **Cytoplasmic vs. Nuclear Intermediate Filaments -- General Functions/Roles of Each** - **Cytoplasmic intermediate filaments:** These filaments provide structural support, flexibility, and resistance to mechanical stress, especially in cells subject to stretching or compression (like epithelial cells). Different types of cytoplasmic intermediate filaments (such as **keratins**, **vimentin**, and **neurofilaments**) are found in various cells. Keratins, for example, are abundant in epithelial cells, forming a network that helps cells withstand physical stress. - **Nuclear intermediate filaments:** Primarily composed of **lamins**, these filaments form a mesh-like structure under the nuclear envelope, known as the nuclear lamina. They provide support to the nucleus, help maintain its shape and regulate DNA replication and transcription by organizing chromatin. **Nature of Intermediate Filament Assembly** Intermediate filaments (IFs) are constructed differently from actin filaments and microtubules. Their assembly is based on the staggered coiling of fibrous proteins: 1. Two monomers wrap around each other to form a coiled-coil dimer. 2. Two dimers align in an antiparallel arrangement to form a tetramer. 3. Tetramers align end-to-end to form protofilaments. 4. Several protofilaments bundle and twist to create a rope-like intermediate filament. Unlike actin and microtubules, IFs do not have polarity (no "+" or "-" end) and do not require ATP or GTP for their assembly. **Regulation of Intermediate Filament Assembly/Disassembly** Intermediate filament assembly is regulated mainly by phosphorylation and dephosphorylation. Phosphorylation usually leads to the disassembly of intermediate filaments, while dephosphorylation promotes reassembly. For instance, during cell division, nuclear lamins are phosphorylated, causing the nuclear envelope to break down for mitosis. After cell division, dephosphorylation triggers lamins to reform the nuclear lamina. **Microtubules -- Composition, Structure, Polarity of Filaments in Cells** - **Composition:** Microtubules are composed of alpha- and beta-tubulin heterodimers. These dimers assemble in a head-to-tail fashion to create a protofilament. - **Structure:** Thirteen protofilaments arrange themselves in a circular pattern to form a hollow, tube-like structure approximately 25 nm in diameter. - **Polarity:** Microtubules have distinct polarity, with a "+" end (fast-growing, where beta-tubulin is exposed) and a "-" end (slow-growing, where alpha-tubulin is exposed). In cells, the "-" end is often anchored at the microtubule-organizing center (MTOC), such as the **centrosome**, while the "+" end extends toward the **cell** **periphery**. **Dynamic Instability -- Meaning of This** Dynamic instability refers to the continuous cycle of growth and shrinkage at the "+" end of a microtubule. GTP-bound tubulin adds to the growing end, but when GTP hydrolyzes to GDP, tubulin becomes less stable and may dissociate, causing rapid shrinkage (also known as a "catastrophe"). The cycle can switch between phases of growth and shrinkage, allowing microtubules to explore cellular space quickly. **Regulation of Microtubules at the Growing End** Proteins like +TIPs (plus-end tracking proteins) stabilize the growing "+" end and help control microtubule dynamics. Additionally, GTP-tubulin serves as a cap at the growing end, maintaining stability. When this cap is lost, microtubules depolymerize rapidly. **How Microtubules Are Nucleated** Microtubules nucleate from the MTOC, with gamma-tubulin complexes in the centrosome playing a key role. Gamma-tubulin forms a ring complex that provides a template for the addition of alpha- and beta-tubulin dimers, initiating microtubule growth. **Organization and Types of Microtubules in the Mitotic Spindle** The mitotic spindle is composed of three main types of microtubules: 1. **Astral microtubules:** Extend outward from the centrosome, anchoring the spindle to the cell membrane. 2. **Kinetochore microtubules:** Attach to the chromosomes' kinetochores and pull chromatids apart during anaphase. 3. **Interpolar microtubules:** Overlap at the center of the cell and push the spindle poles apart, helping maintain spindle structure. **Spindle and Chromosome Events in Metaphase and Anaphase** - **Metaphase:** Chromosomes align at the metaphase plate, and kinetochores of each sister chromatid attach to kinetochore microtubules from opposite spindle poles. - **Anaphase:** Kinetochore microtubules shorten, pulling sister chromatids toward opposite poles (Anaphase A). At the same time, interpolar microtubules lengthen, pushing the spindle poles apart (Anaphase B). **Microtubule-Based Motor Proteins -- How They Move, Directionality of the Two Types** - **Dynein:** Moves toward the "-" end of microtubules (toward the cell center). It is involved in transporting cargoes inward, like vesicles and organelles. - **Kinesin:** Moves toward the "+" end of microtubules (toward the cell periphery). It typically carries cargoes outward. Both motor proteins move by hydrolyzing ATP, causing conformational changes in their structures that enable them to "walk" along the microtubule. **Actin Cytoskeleton -- Where It Is Found in the Cell** The actin cytoskeleton is present throughout the cell but is concentrated near the cell cortex (just beneath the plasma membrane), where it provides structural support and plays a role in cell shape, adhesion, and movement. **Differences of Actin Organization in Filopodia, Lamellipodia, and Cortical Actin in Motile Cells** - **Filopodia:** Thin, finger-like protrusions formed by tightly bundled, parallel actin filaments. They help cells sense the environment and navigate. - **Lamellipodia:** Flat, sheet-like structures with branched networks of actin filaments that allow the cell to push forward in a crawling motion. - **Cortical actin:** Forms a dense network under the cell membrane, stabilizing cell shape and helping in cellular contractions during movement. **Regulation of Actin Assembly/Disassembly Dynamics in Filaments** Actin dynamics are regulated by actin-binding proteins that control nucleation, elongation, capping, and severing. ATP-actin adds to the "+" end, while ADP-actin is more likely to disassemble from the "-" end. Proteins like cofilin can bind ADP-actin, promoting disassembly, while profilin can enhance ATP-actin binding for growth. **Actin Interactions with Myosin Motor Proteins -- Muscle Cells and During Cytokinesis** In muscle cells, actin filaments interact with myosin to create contractile forces essential for muscle contraction. During cytokinesis, the contractile ring composed of actin and myosin II forms at the cell equator and constricts to split the cell into two daughter cells. **Muscle Cell Organization** Muscle cells (fibers) are organized into bundles of myofibrils, which consist of repeating units called sarcomeres. Sarcomeres contain actin (thin) filaments and myosin (thick) filaments organized in a regular, overlapping pattern that gives muscle its striated appearance. **Mechanism Underlying Contraction and Relaxation of Muscle Cells** Muscle contraction is triggered when calcium ions bind to troponin, causing tropomyosin to move and expose myosin-binding sites on actin. Myosin heads attach to actin, perform a power stroke by bending, and pull actin filaments toward the sarcomere center. This shortens the sarcomere, resulting in contraction. Relaxation occurs when calcium ions are removed, allowing tropomyosin to block the myosin-binding sites on actin again, stopping contraction. These explanations provide a comprehensive overview of each topic related to cytoskeletal dynamics in cells. **LECTURE (Chapter 19) --- Transmission Genetics** **Mendel's Contributions** Gregor Mendel, an Austrian monk, is known as the \"father of genetics\" for his pioneering work on inheritance in pea plants. Mendel's contributions include: - **Law of Segregation:** Each organism inherits two alleles for each trait, one from each parent. These alleles segregate during gamete formation, ensuring each gamete only carries one allele for each trait. - **Law of Independent Assortment:** Alleles for different traits are inherited independently if they are located on different chromosomes. - **Dominant and Recessive Traits:** Mendel observed that certain traits consistently appeared in one generation but could skip generations when recessive. These findings laid the groundwork for the understanding of dominance and recessiveness. **Dominance and Recessiveness in Diploid Sexually Reproducing Organisms** In diploid organisms, each individual has two alleles for each gene, one from each parent. If one allele (A) is dominant and the other (a) is recessive: - **Dominant allele (A)**: This allele can mask the expression of a recessive allele in a heterozygous individual (Aa). If a dominant allele is present, it typically determines the phenotype. - **Recessive allele (a)**: This allele only influences the phenotype if both alleles are recessive (aa). **Genotype and phenotype differences:** - **AA (homozygous dominant):** Displays the dominant phenotype. - **Aa (heterozygous):** Displays the dominant phenotype, as the A allele masks the recessive a allele. - **aa (homozygous recessive):** Displays the recessive phenotype, as no dominant allele is present to mask it. **Meanings of the Terms Locus and Allele** - **Locus:** The specific location or position of a gene on a chromosome. Each gene occupies a particular locus. - **Allele:** Different versions or forms of a gene that may exist at a specific locus. For instance, in the gene for eye color, one allele may code for brown eyes and another for blue eyes. **Penetrance and Expressivity** - **Penetrance:** The proportion of individuals with a specific genotype that actually displays the associated phenotype. If a trait has incomplete penetrance, not all individuals with the genotype will show the phenotype. - **Expressivity:** The degree or intensity with which a genotype is expressed in the phenotype. Variations in expressivity mean that individuals with the same genotype can display different severities or manifestations of the phenotype. **Difference Between Mitosis and Meiosis** - **DNA replication:** Occurs once before both mitosis and meiosis during the S phase of the cell cycle. - **Division of homologs and sister chromatids:** - **Mitosis:** Involves a single division where sister chromatids separate, resulting in two genetically identical daughter cells. - **Meiosis:** Consists of two divisions: - **Meiosis I:** Homologous chromosomes (each with sister chromatids) separate. - **Meiosis II:** Sister chromatids separate, leading to four genetically unique haploid cells. **Meaning of Independent Assortment of Traits** Independent assortment refers to the random distribution of chromosome pairs (and thus genes) into gametes during meiosis. This means alleles for different traits segregate independently if they are on different chromosomes. However, traits do not assort independently if the genes are located close together on the same chromosome (linkage). **How Recombination Frequency Relates to Genetic Map Distance** Recombination frequency measures how often two genes recombine due to crossing over during meiosis. The higher the recombination frequency, the farther apart the genes are on a chromosome. Recombination frequency is used to create genetic maps, where a 1% recombination frequency roughly equals 1 centimorgan (cM), representing genetic distance. **Definitions of SNPs and Their Use in GWAS Studies** - **Single Nucleotide Polymorphisms (SNPs):** These are variations in a single nucleotide at a specific position in the genome, commonly occurring in the population. SNPs can be associated with particular traits or diseases. - **GWAS (Genome-Wide Association Studies):** GWAS use SNPs to identify genetic variants associated with specific traits or diseases. By comparing SNP patterns across many individuals, researchers can identify genetic regions that contribute to disease risk. **Quantitative Traits and Their Genetic Basis** Quantitative traits are controlled by multiple genes and influenced by environmental factors, resulting in a continuous range of phenotypes. Examples include height, weight, and skin color. In humans, height is a classic quantitative trait with contributions from many genes and environmental factors. **Dihybrid Cross: Expected 9:3:3:1 Ratio and Deviations** In a dihybrid cross where both parents are heterozygous for two traits (Aa Bb x Aa Bb), the offspring typically show a 9:3:3:1 phenotypic ratio: - **9** with both dominant traits. - **3** with one dominant trait and the other recessive. - **3** with the other dominant trait and one recessive. - **1** with both recessive traits. If the observed ratio deviates, it could indicate: - **Linkage:** Genes are located on the same chromosome and inherited together. - **Epistasis:** Interaction between genes that alters the expected phenotypic ratios. - **Lethal alleles:** Some genotypes might be lethal, reducing certain phenotypes. **Meaning of Aneuploidy** Aneuploidy is a condition where cells have an abnormal number of chromosomes, resulting from nondisjunction during meiosis. Common examples in humans include trisomy 21 (Down syndrome), where there is an extra copy of chromosome 21. **Value of Genetic Recombination and Crossing Over** Genetic recombination (crossing over) occurs during meiosis I when homologous chromosomes exchange segments. This creates new combinations of alleles, increasing genetic diversity. This diversity is advantageous for populations as it allows for adaptation and survival in changing environments. This explanation covers each concept with detail to provide a thorough understanding of genetics principles. **LECTURE (Chapter 16) --- Signaling** **Steps of Signaling** 1. **Ligand/Receptor Interaction:** A signaling molecule (ligand) binds to a specific receptor on the cell surface or inside the cell. This receptor is typically a protein that changes shape or activates upon ligand binding. 2. **Signal Transduction:** The receptor transmits the signal inside the cell through a cascade of molecular interactions. This often involves secondary messengers or phosphorylation events, which alter the activity of various intracellular proteins. 3. **Signal Amplification:** In many pathways, a single ligand binding event can trigger a chain reaction, activating multiple downstream molecules, which amplifies the initial signal. 4. **Effectors:** These are the molecules that directly bring about the cellular response, such as transcription factors that initiate gene expression or enzymes that modify cell behavior. 5. **Termination of Signaling:** The signal is terminated to prevent overactivation. Mechanisms for termination include: - **Dephosphorylation** of Receptor Tyrosine Kinases (RTKs). - **Re-association** of heterotrimeric G proteins after GTP hydrolysis. - **Endocytosis** and degradation of GPCRs and RTKs to remove receptors from the cell surface and prevent further signaling. **Types of Signaling** 1. **Endocrine:** Involves hormones released into the bloodstream, where they travel to distant target cells. This type is characterized by long-range signaling. 2. **Paracrine:** Signaling molecules affect nearby cells. Paracrine signaling is short-range, as the molecules are typically rapidly degraded. 3. **Neuronal:** Uses neurotransmitters to transmit signals along nerve cells or across synapses to neighboring cells. This form of signaling is fast and highly localized. 4. **Contact-Dependent:** Requires direct contact between signaling and target cells. Molecules on the surface of one cell interact with receptors on the adjacent cell. **Steroid Hormone Signaling Mechanisms** Steroid hormones (e.g., cortisol, estrogen) are hydrophobic and can pass through the cell membrane. They bind to intracellular receptors (often in the cytoplasm or nucleus) that directly modulate gene expression by acting as transcription factors. This is distinct from many other signaling types, where receptors are membrane-bound, and the signal transduction involves secondary messengers. **Processes Controlled by TOR Kinase** TOR (Target of Rapamycin) kinase is a major regulator of cell growth and metabolism. It responds to nutrient levels, growth factors, and energy status to control: - Protein synthesis - Autophagy (self-digestion in response to low nutrients) - Lipid synthesis - Cell growth and proliferation **Structure and Function of Integrins** Integrins are transmembrane receptors composed of alpha and beta subunits (heterodimers). They mediate cell adhesion to the extracellular matrix (ECM) and send signals into the cell to modulate cytoskeletal organization, survival, and proliferation. Integrins can cluster upon binding to the ECM, initiating signaling cascades that influence cellular responses. **GPCRs and Heterotrimeric G Proteins** - **GTP Binding Proteins:** In a heterotrimeric G protein, the alpha subunit binds GTP, while the beta and gamma subunits often remain together. - **Regulation:** G proteins are activated when GTP binds to the alpha subunit (GTP-bound = "on") and are inactivated when GTP is hydrolyzed to GDP (GDP-bound = "off"). - **Association with Other Proteins:** GPCRs activate G proteins by promoting the exchange of GDP for GTP on the alpha subunit, which then dissociates from the beta-gamma dimer to signal downstream effectors. **GPCR Signaling Pathways** 1. **cAMP Pathway:** - Activation: A GPCR activates the G protein, which then activates adenylyl cyclase. - cAMP Production: Adenylyl cyclase converts ATP to cAMP. - Role of cAMP: Acts as a second messenger to activate Protein Kinase A (PKA), leading to downstream effects. - Termination: Phosphodiesterase breaks down cAMP to terminate the signal. 2. **Phospholipase C (PLC) Pathway:** - Activation: GPCRs can activate PLC, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and DAG. - IP3 and DAG Roles: IP3 releases Ca2+ from the ER, while DAG activates Protein Kinase C (PKC), initiating other downstream effects. **RTK Signaling to Ras and MAPK Cascades** - **RTK Activation:** Ligand binding causes RTKs to dimerize and autophosphorylate, creating docking sites for proteins. - **Ras Activation:** Adapter proteins link RTKs to Ras, a small GTPase. Ras binds GTP and activates downstream MAPK (Mitogen-Activated Protein Kinase) cascades. - **MAPK Cascade:** This cascade (e.g., Ras → Raf → MEK → ERK) transmits signals to the nucleus, often resulting in changes in gene expression. **Integrins Signaling to the Cytoskeleton** Upon binding to the ECM, integrins recruit proteins like focal adhesion kinase (FAK) and Src, which activate signaling pathways that regulate the cytoskeleton, promoting cell migration, shape changes, and proliferation. **Calmodulin** - **Activation:** Calmodulin is activated by binding to calcium ions (Ca2+). - **Role:** Once activated, calmodulin binds to and activates various target proteins, including kinases and phosphatases, regulating cellular processes like muscle contraction, cell division, and metabolism. **Nature of Different Signaling Pathways** - **GTP/GDP Binding:** Small GTPases like Ras are "on" when bound to GTP and "off" when bound to GDP, switching due to regulatory proteins like GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins). - **Phosphorylation/Kinase Cascades:** Many pathways involve kinase cascades, where each kinase phosphorylates and activates another in sequence. For example, the MAPK cascade transmits signals through successive phosphorylation events. - **Phosphoproteins in Signal Transduction:** Phosphorylated proteins, such as MAPK, serve as intermediates in signal transduction, passing signals to downstream effectors or altering cellular activities by modulating transcription factors. **Exam 4** ION AND SOLUTE TRANSPORT **1. Ion and Solute Transport** - **Ions and solutes** move across cell membranes via **channels** or **transporters**, depending on their properties and the membrane\'s selective permeability. **2. Selective Permeability of Membranes** - Membranes are **selectively permeable**, meaning some molecules can pass through while others cannot: - **Pass:** Small nonpolar molecules (O₂, CO₂), small polar molecules (water via aquaporins). - **Don't Pass:** Large polar molecules (glucose) and charged ions (Na⁺, K⁺) unless specific channels or transporters are available. **3. Channels vs. Transporters** - **Channels:** - Form pores in the membrane. - Allow specific ions/molecules to pass through **passively**. - Faster than transporters. - **Transporters:** - Undergo conformational changes to move molecules. - Can be **active** (use energy) or **passive** (no energy). **4. Active vs. Passive Transport** - **Passive Transport:** - Molecules move **down their concentration gradient** (high → low). - No energy required. - Includes **diffusion** and **facilitated diffusion**. - **Active Transport:** - Molecules move **against their concentration gradient** (low → high). - Requires energy, typically ATP or an electrochemical gradient. **5. Symport vs. Antiport** - **Symport:** - Moves two substances in the **same direction**. - Example: Sodium-glucose transporter (moves glucose into cells with Na⁺). - **Antiport:** - Moves two substances in **opposite directions**. - Example: Sodium-potassium pump (Na⁺ out, K⁺ in). **6. Symport and Antiport in Gut Epithelial Cells** - **Glucose Uptake:** - **Symport:** Glucose enters cells with Na⁺ via the sodium-glucose symporter (SGLT) on the apical membrane. - **Glucose Release:** - **Passive Transport:** Glucose exits into the bloodstream through a glucose transporter (GLUT) on the basolateral membrane. **7. Electrochemical Gradient** - The **electrochemical gradient** is the combined effect of: - **Concentration Gradient:** Difference in ion concentration. - **Electrical Gradient:** Difference in charge across the membrane. - Drives ion movement (e.g., Na⁺ wants to enter cells because of its high extracellular concentration and the negative charge inside cells). **8. Energy in Active Transport** - Pumps like the **Na⁺/K⁺ ATPase** use ATP to move ions against their electrochemical gradients: - 3 Na⁺ out of the cell. - 2 K⁺ into the cell. - This maintains the resting potential and ion gradients. **9. Ligands and Channel Gating** - Channels open in response to: - **Ligands:** Molecules that bind to the channel (e.g., neurotransmitters like acetylcholine). - **Voltage Changes:** Voltage-gated channels respond to changes in membrane potential. - **Mechanical Forces:** Mechanosensitive channels. **10. Na⁺/K⁺ ATPase** - **What It Is:** - A **carrier**, not a channel. - Uses ATP to transport 3 Na⁺ out and 2 K⁺ into the cell. - **What It Does:** - Maintains the resting potential. - Helps regulate cell volume and ion gradients. **11. Action Potentials** - **Initiation and Propagation:** - Triggered by depolarization (membrane becomes less negative). - Major channel: **Voltage-gated Na⁺ channels**. - Steps: 1. Stimulus opens some Na⁺ channels. 2. Na⁺ rushes in, further depolarizing the membrane. 3. Threshold reached → action potential generated. - **Propagation:** Depolarization spreads to adjacent areas, opening more Na⁺ channels. **12. Resetting Resting Potential** - After an action potential: - **Voltage-gated K⁺ channels** open → K⁺ exits the cell, repolarizing the membrane. - **Na⁺/K⁺ ATPase** restores the ion gradients. **13. Voltage-Gated Na⁺ Channel States** - **Open:** During depolarization, Na⁺ flows in. - **Inactive:** Quickly after opening, the channel blocks ion flow (refractory period). - **Closed:** Returns to resting state, ready to open again when stimulated. **14. Membrane Voltage and Ion Disposition** - Inside the cell: - **K⁺ is high**. - **Na⁺ and Ca²⁺ are low**. - Resting potential: **-70 mV**, due to K⁺ leak channels and Na⁺/K⁺ ATPase. **15. Inhibitory vs. Excitatory Neurons** - **Excitatory Neurons:** - Depolarize the membrane (e.g., by opening Na⁺ channels). - Increase likelihood of firing action potentials. - **Inhibitory Neurons:** - Hyperpolarize the membrane (e.g., by opening Cl⁻ or K⁺ channels). - Reduce likelihood of firing action potentials. **16. Voltage-Gated Ca²⁺ Channels in Synaptic Vesicle Docking** - **Role in Neuron Communication:** 1. Action potential reaches the axon terminal. 2. **Voltage-gated Ca²⁺ channels** open. 3. Ca²⁺ enters the cell. 4. Ca²⁺ triggers synaptic vesicles to dock at the membrane and release neurotransmitters via **exocytosis**. - **Key Terms:** 1. **Synaptic Vesicle Docking:** Vesicles containing neurotransmitters align with the presynaptic membrane. 2. **Regulated Exocytosis:** Neurotransmitters are released into the synaptic cleft in response to Ca²⁺. MITOCHONDRIA AND CHLOROPLASTS **1. Chemiosmotic Coupling** - **Definition:** - The process of generating ATP by using the energy stored in a **H⁺ gradient** across a membrane. - It couples two events: 1. **Electron Transport Chain (ETC):** Creates a proton (H⁺) gradient. 2. **ATP Synthase Activity:** Uses the gradient to synthesize ATP. **2. H⁺ Gradient** - **Where Does It Come From?** - In mitochondria: - Electrons flow through the **ETC** (on the inner mitochondrial membrane), transferring energy to pump H⁺ ions from the **matrix** to the **intermembrane space**. - In chloroplasts: - During the **light reactions** in photosynthesis, the **thylakoid membrane** pumps H⁺ ions into the **thylakoid lumen**. - **Purpose of the H⁺ Gradient:** - Creates an **electrochemical gradient** (difference in charge and concentration) that drives ATP synthesis when H⁺ flows back through **ATP synthase**. - In mitochondria: - Powers ATP production to support cellular processes. - In chloroplasts: - Produces ATP to power sugar synthesis during the Calvin cycle. **3. Electron Transport Complexes and Chemiosmotic Coupling** - **Purpose of the ETC:** - Series of protein complexes that transfer electrons from energy carriers (NADH, FADH₂ in mitochondria or light-excited electrons in chloroplasts) to a final acceptor. - The energy released during electron transfer powers **H⁺ pumping** across the membrane. - **Complexes in Mitochondria:** - - - - - **Complexes in Chloroplasts:** - Proteins in the **thylakoid membrane** (e.g., photosystem II, cytochrome b₆f) transfer electrons and pump H⁺ ions. **4. Structures and Membranes in Mitochondria vs. Chloroplasts** **Feature** **Mitochondria** **Chloroplasts** --------------------------- ----------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------- **Outer Membrane** Permeable to small molecules via porins. Permeable, contains porins. **Inner Membrane** Impermeable to most ions, houses the ETC and ATP synthase. Impermeable to ions, houses light reactions. **Internal Compartments** **Matrix:** Site of Krebs cycle and low H⁺ concentration. **Intermembrane Space:** High H⁺ concentration. **Stroma:** Site of Calvin cycle and low H⁺ concentration. **Thylakoid Lumen:** High H⁺ concentration. **Energy Source** Energy from oxidation of glucose/fatty acids. Energy from sunlight (photosynthesis). **5. ATP Synthase** - **How It Functions:** - Located in the **inner mitochondrial membrane** or **thylakoid membrane**. - As H⁺ ions flow down their gradient (from intermembrane space → matrix or thylakoid lumen → stroma), the flow spins ATP synthase. - This mechanical energy converts ADP + Pi → ATP. - **Where Is ATP Made?** - Mitochondria: ATP is produced in the **matrix**. - Chloroplasts: ATP is produced in the **stroma**. - **How ATP Is Made Available:** - Mitochondria: - ATP is transported out of the matrix via **ATP/ADP translocase** to the cytoplasm, where it powers cellular work. - Chloroplasts: - ATP stays in the stroma to fuel the Calvin cycle for sugar synthesis. **6. Summary: How Energy Powers ATP Production** 1. **In Mitochondria:** - Electrons from NADH/FADH₂ move through the ETC. - Energy from electron flow pumps H⁺ into the intermembrane space. - H⁺ flows back into the matrix through ATP synthase, driving ATP production. 2. **In Chloroplasts:** - Light excites electrons in photosystem II. - The ETC pumps H⁺ into the thylakoid lumen. - H⁺ flows back into the stroma through ATP synthase, producing ATP. CELL CYCLE AND ITS REGULATION, CELL DEATH MECHANISMS **1. Phases of the Cell Cycle and General Events** 1. **G₁ Phase (Gap 1):** - Cell grows, synthesizes proteins and organelles, and prepares for DNA replication. - Decision to proceed (commit to division) or enter G₀ (resting phase). 2. **S Phase (Synthesis):** - DNA replication occurs, resulting in two identical sister chromatids for each chromosome. 3. **G₂ Phase (Gap 2):** - Cell prepares for mitosis by synthesizing proteins and repairing any DNA damage. 4. **M Phase (Mitosis):** - Divided into **prophase**, **metaphase**, **anaphase**, and **telophase**. - Ends with **cytokinesis** (division of cytoplasm into two daughter cells). 5. **G₀ Phase:** - Resting state where cells are metabolically active but do not divide (e.g., nerve cells). **2. Cdk-Complexes (Cyclin-Dependent Kinases)** - **Components:** 1. **Cyclin:** Regulatory protein whose levels fluctuate during the cell cycle. 2. **Cdk (Cyclin-Dependent Kinase):** Enzyme that phosphorylates target proteins to drive the cell cycle forward. - **Regulation of Cyclins and Cdks:** 3. **Cyclins:** - Synthesized and degraded at specific stages of the cell cycle. - Example: M-cyclin accumulates during G₂ and is degraded after mitosis. 4. **Cdks:** - Activated by cyclin binding. - Regulated by **phosphorylation**: - Inhibitory phosphorylation (e.g., Wee1 kinase adds inhibitory phosphate). - Activating phosphorylation (e.g., Cdc25 phosphatase removes the inhibitory phosphate). **3. Rapid Activation of M-Cdks** - **How it happens:** 1. **Accumulation of M-cyclin:** M-cyclin levels rise in G₂. 2. **Phosphorylation Events:** - Inhibitory phosphorylation by **Wee1 kinase** keeps M-Cdk inactive. - Activating phosphorylation by **Cdc25 phosphatase** removes the inhibitory phosphate. 3. **Positive Feedback Loop:** - Active M-Cdk activates more Cdc25 and inhibits Wee1, leading to a rapid surge in M-Cdk activity. **4. Targets of Cdk Complexes** 1. **Nuclear Lamins:** - Phosphorylated by M-Cdk during prophase, causing lamins to disassemble and the nuclear envelope to break down. 2. **Condensins and Cohesins:** - **Condensins:** Help condense chromosomes for mitosis. - **Cohesins:** Hold sister chromatids together until anaphase. 3. **Integrins:** - Regulated by Cdks to allow changes in cell adhesion during division. **5. Role of p53 and p21 in DNA Damage Response** - **p53:** - A tumor suppressor protein activated by DNA damage. - Promotes transcription of **p21**, a Cdk inhibitor. - Prevents progression from G₁ to S phase, allowing time for DNA repair or triggering apoptosis if damage is too severe. - **p21:** - Inhibits G₁/S-Cdk and S-Cdk, halting the cell cycle. **6. Functions of Mitogens** - **What They Do:** - Extracellular signals (growth factors) that stimulate cell division. - Bind to **receptor tyrosine kinases (RTKs)** to activate signaling pathways (e.g., Ras → MAPK pathway). - Promote the synthesis of cyclins (e.g., G₁-cyclin) needed for cell cycle progression. - **Role in Rb Regulation:** - **Rb (Retinoblastoma protein):** Inhibits transcription factors (like E2F) required for S phase entry. - **Mitogens activate G₁/S-Cdk**, which phosphorylates Rb, inactivating it and freeing E2F. - E2F promotes transcription of genes required for DNA replication and cell division. **7. Apoptosis (Programmed Cell Death)** - **Key Players:** 1. **Caspases:** - Proteins that execute apoptosis by cleaving target proteins. - **Initiator Caspases:** Activated first (e.g., Caspase-9). - **Effector Caspases:** Execute cell death (e.g., Caspase-3). 2. **Cytochrome c:** - Released from mitochondria into the cytoplasm during apoptosis. - Activates **Apaf-1**, forming the apoptosome to activate caspases. 3. **Bcl-2 Family:** - **Anti-apoptotic:** Prevent cytochrome c release (e.g., Bcl-2). - **Pro-apoptotic:** Promote cytochrome c release (e.g., Bax, Bak). **8. FACs Analysis (Fluorescence-Activated Cell Sorting)** - **What It Does:** - Measures DNA content in cells to analyze their position in the cell cycle. - **How It Works:** - Cells are stained with a DNA-binding dye. - Flow cytometry measures fluorescence intensity: - **G₁ Phase:** Low DNA content. - **S Phase:** Intermediate DNA content (replicating DNA). - **G₂/M Phase:** High DNA content. TISSUES AND STEM CELLS **1. General Mechanisms of Cell Differentiation and Tissue Formation** - **Differentiation:** - Process by which unspecialized cells (stem cells) become specialized in structure and function. - **Mechanisms:** 1. **Gene Expression:** - Differential expression of genes leads to specialized proteins for distinct cell types. 2. **Cell Signaling:** - Signals from neighboring cells guide differentiation (e.g., growth factors, morphogens). 3. **Epigenetics:** - Chromatin structure changes (e.g., methylation, histone modification) lock cells into specific fates. - **Tissue Formation:** 1. Involves **cell adhesion**, **cell-cell signaling**, and interactions with the **extracellular matrix (ECM)**. **2. Stem Cells** - **Definition:** - Undifferentiated cells capable of self-renewal and differentiation into various cell types. - **Potency Types:** - - - - - **Induced Pluripotent Stem Cells (iPSCs):** - Created by reprogramming differentiated cells using transcription factors like **Oct4**, **Sox2**, **Klf4**, and **c-Myc**. - **Embryonic Stem Cells (ESCs):** - Derived from the **inner cell mass of blastocysts**. - **Stem Cell Niche:** - Microenvironment providing signals (e.g., ECM, growth factors) to maintain stem cells or guide differentiation. **3. Extracellular Matrix (ECM)** - **Components:** - **Plants:** - **Cellulose:** Structural polysaccharide synthesized by cellulose synthase in the plasma membrane. - Provides rigidity to cell walls. - **Animals:** - **Collagen:** Major fibrous protein, provides tensile strength. - Collagen fibril assembly occurs extracellularly with help from enzymes like **lysyl oxidase**. - **Proteoglycans:** Hydrated molecules providing compressive resistance. - **Fibronectin/Laminin:** Adhesive proteins connecting cells to the ECM. - **Control of Deposition and Assembly:** - Enzymes like **cellulose synthase (plants)** or **procollagen peptidase (animals)** regulate assembly. **4. Integrins and ECM Attachment** - **Integrins:** - Transmembrane receptors that link the ECM to the cytoskeleton. - Bind to ECM components like **fibronectin** and **collagen** outside the cell. - Inside the cell, connect to the **actin cytoskeleton** via adaptor proteins (e.g., talin, vinculin). **5. Cell Junctions in Epithelial Sheets** 1. **Tight Junctions:** - **Purpose:** Prevent leakage between cells and maintain polarity. - **Proteins:** Claudins, occludins. - Link to **actin cytoskeleton**. 2. **Adherens Junctions:** - **Purpose:** Provide mechanical support by connecting actin filaments between cells. - **Proteins:** Cadherins, catenins. 3. **Desmosomes:** - **Purpose:** Connect intermediate filaments between cells for strong adhesion. - **Proteins:** Desmogleins, desmocollins. 4. **Gap Junctions:** - **Purpose:** Allow direct communication between cells via small molecules. - **Proteins:** Connexins. 5. **Hemidesmosomes:** - **Purpose:** Anchor cells to the ECM. - **Proteins:** Integrins, linked to intermediate filaments. **6. Cadherins** - **Function:** - Mediate cell-cell adhesion in a calcium-dependent manner. - **Requirements for Activity:** - Binding of extracellular calcium stabilizes cadherin structure. - Link to cytoskeleton through adaptor proteins like **catenins**. **7. Plasmodesmata** - **Definition:** - Channels in plant cell walls that allow the exchange of molecules and ions between adjacent cells. - **Function:** - Facilitate communication and transport of nutrients, signaling molecules, and RNA. **8. Cancer** - **Multi-Hit Mutation Model:** - Cancer arises from the accumulation of mutations in genes regulating cell growth and division. - **Environmental Inputs:** Carcinogens (e.g., UV radiation, smoking). - **Genetic Inputs:** Inherited mutations (e.g., BRCA1). - **Oncogenes:** - **Definition:** Mutated or overactive forms of genes that promote cell division. - **Example:** **Ras** -- a GTPase that becomes constitutively active, driving uncontrolled growth. - **Tumor Suppressors:** - **Definition:** Genes that inhibit cell division or promote apoptosis. Mutations lead to loss of function. - **Example:** **p53** -- triggers cell cycle arrest or apoptosis in response to DNA damage.