Basic organization and functional features of the eyespot:

• Structure: The eyespot, also known as the stigma or the paraflagellar body, is a specialized organelle found in the flagellated cells of organisms like Chlamydomonas. It contains photoreceptor pigments, such as rhodopsin, which are sensitive to light.

• Function: The eyespot allows the organism to detect the direction of light. This information is used for phototaxis, the movement toward or away from light.

Structure & function of channelrhodopsin and voltage-gated Na channel:

• Channelrhodopsin: It is a light-sensitive ion channel found in the eyespot of Chlamydomonas. When activated by light, it allows the passage of ions across the cell membrane, leading to changes in membrane potential and ultimately affecting the cell's behavior.

• Voltage-gated Na channel: Found in neurons, it is responsible for generating action potentials by allowing the influx of sodium ions into the cell when the membrane potential reaches a certain threshold.

Basics of how an action potential is generated:

• Resting State: The cell membrane is polarized, with a negative charge inside and positive charge outside.

• Depolarization: Stimulus triggers the opening of ion channels, allowing sodium ions to rush into the cell, reversing the polarity temporarily (depolarization).

• Repolarization: Potassium channels open, allowing potassium ions to leave the cell, restoring the negative charge inside (repolarization).

Channelrhodopsin and photoisomerization of retinal:

• Channelrhodopsin contains retinal pigment, which undergoes photoisomerization upon light absorption, leading to a conformational change in the opsin protein and subsequent ion channel opening.

• In the human eye, rhodopsin undergoes similar photoisomerization, resulting in a change in the shape of the opsin protein, initiating the visual signal transduction pathway.

Key similarities and differences between channelrhodopsin and rhodopsin:

• Similarities: Both channelrhodopsin and rhodopsin contain a retinal pigment bound to an opsin protein, and both undergo photoisomerization upon light absorption.

• Differences: Channelrhodopsin functions as an ion channel, while rhodopsin is involved in visual signal transduction. Channelrhodopsin is found in the eyespot of Chlamydomonas, while rhodopsin is found in the photoreceptor cells of the human eye.

Optogenetics and expressing Chlamy opsin in brain cells:

• Optogenetics involves genetically modifying neurons to express light-sensitive proteins, such as Chlamydomonas channelrhodopsin. This allows researchers to control neuronal activity using light stimulation, enabling the study of neural circuits and behavior.

Mental Floss: Phototaxis:

• Loss of Phototactic Ability: A mutation that disrupts the structure or function of the eyespot or its associated proteins, such as channelrhodopsin, could cause cells to lose their ability to detect light direction and exhibit phototaxis.

• Movement Away from Light: Cells might move away from light to avoid harmful conditions, such as excessive light exposure or high temperatures, or to seek optimal environmental conditions for growth and survival.

Class Outcomes:

1. Major structural features of a Chlamydomonas cell:

   • Chlamydomonas cells have a single cup-shaped chloroplast, two flagella, a nucleus, a large central vacuole, and a cell wall.

2. Features of Chlamy grown in the lab:

   • Chlamydomonas grown in the lab requires a nutrient-rich medium containing inorganic salts, vitamins, and a carbon source like acetate or glucose.

3. Difference between Macronutrient and Micronutrient:

   • Macronutrients are required in large quantities by organisms and include elements such as carbon, nitrogen, and phosphorus. Micronutrients are required in smaller quantities and include elements like iron, zinc, and manganese.

4. Why Chlamydomonas and humans need phosphate (PO4) and iron (Fe):

   • Phosphate is essential for DNA, RNA, and ATP synthesis, while iron is required for chlorophyll and hemoglobin synthesis, electron transport, and enzyme function.

5. Growth and doubling time:

   • Chlamydomonas grows exponentially, doubling in population size in approximately 10 hours at 25°C. This means that the population doubles every 10 hours under optimal growth conditions.

6. Microbial growth curve:

   • The growth curve shows three phases: lag phase (adaptation to new environment), exponential phase (rapid growth), and stationary phase (nutrient depletion and waste accumulation).

7. Chlamydomonas phylogeny and relationship to plants and animals:

   • Chlamydomonas is a green alga and is evolutionarily related to land plants. It shares a common ancestor with plants but is more closely related to animals than to plants.

8. Explanation for why Chlamydomonas and humans have flagella but plants do not:

   • The simplest explanation is that Chlamydomonas and humans are both motile organisms that require flagella for movement, while plants are typically non-motile and do not require flagella for locomotion.

9. Basics of cilia structure and ciliopathies:

   • Cilia are composed of microtubules arranged in a 9+2 pattern and are involved in cell motility and signaling. Mutations in cilia-related proteins can cause ciliopathies, which are associated with various human diseases.

10. Distinctions between motile and non-motile flagella:

    • Motile flagella, like those in Chlamydomonas, are involved in movement, while non-motile flagella, like those in sensory cells, are involved in sensory reception.

Mental Floss Questions:

1. Characteristics of Chlamy that make it a "model experimental system":

   • Chlamydomonas is unicellular, haploid, photosynthetic, and genetically tractable, making it an ideal model organism for studying various biological processes.

2. Reason Chlamy stops dividing during stationary phase:

   • Chlamy stops dividing during stationary phase due to nutrient depletion, accumulation of waste products, and changes in environmental conditions.

3. Analysis of 7,476 Chlamy proteins:

   • Proteins common to only Chlamy and humans, only Chlamy and Arabidopsis, and all three species likely reflect shared biological functions and evolutionary relationships between these organisms.

Class Outcomes:

1. Major structural features of a Chlamydomonas cell:

   • Chlamydomonas cells have a single cup-shaped chloroplast, two flagella, a nucleus, a large central vacuole, and a cell wall.

2. Features of Chlamy grown in the lab:

   • Chlamydomonas grown in the lab requires a nutrient-rich medium containing inorganic salts, vitamins, and a carbon source like acetate or glucose.

3. Difference between Macronutrient and Micronutrient:

   • Macronutrients are required in large quantities by organisms and include elements such as carbon, nitrogen, and phosphorus. Micronutrients are required in smaller quantities and include elements like iron, zinc, and manganese.

4. Why Chlamydomonas and humans need phosphate (PO4) and iron (Fe):

   • Phosphate is essential for DNA, RNA, and ATP synthesis, while iron is required for chlorophyll and hemoglobin synthesis, electron transport, and enzyme function.

5. Growth and doubling time:

   • Chlamydomonas grows exponentially, doubling in population size in approximately 10 hours at 25°C. This means that the population doubles every 10 hours under optimal growth conditions.

6. Microbial growth curve:

   • The growth curve shows three phases: lag phase (adaptation to new environment), exponential phase (rapid growth), and stationary phase (nutrient depletion and waste accumulation).

7. Chlamydomonas phylogeny and relationship to plants and animals:

   • Chlamydomonas is a green alga and is evolutionarily related to land plants. It shares a common ancestor with plants but is more closely related to animals than to plants.

8. Explanation for why Chlamydomonas and humans have flagella but plants do not:

   • The simplest explanation is that Chlamydomonas and humans are both motile organisms that require flagella for movement, while plants are typically non-motile and do not require flagella for locomotion.

9. Basics of cilia structure and ciliopathies:

   • Cilia are composed of microtubules arranged in a 9+2 pattern and are involved in cell motility and signaling. Mutations in cilia-related proteins can cause ciliopathies, which are associated with various human diseases.

10. Distinctions between motile and non-motile flagella:

    • Motile flagella, like those in Chlamydomonas, are involved in movement, while non-motile flagella, like those in sensory cells, are involved in sensory reception.

Mental Floss Questions:

1. Characteristics of Chlamy that make it a "model experimental system":

   • Chlamydomonas is unicellular, haploid, photosynthetic, and genetically tractable, making it an ideal model organism for studying various biological processes.

2. Reason Chlamy stops dividing during stationary phase:

   • Chlamy stops dividing during stationary phase due to nutrient depletion, accumulation of waste products, and changes in environmental conditions.

3. Analysis of 7,476 Chlamy proteins:

   • Proteins common to only Chlamy and humans, only Chlamy and Arabidopsis, and all three species likely reflect shared biological functions and evolutionary relationships between these organisms.

Class Outcomes:

1. Major structural features of a Chlamydomonas cell:

   • Chlamydomonas cells have a single cup-shaped chloroplast, two flagella, a nucleus, a large central vacuole, and a cell wall.

2. Features of Chlamy grown in the lab:

   • Chlamydomonas grown in the lab requires a nutrient-rich medium containing inorganic salts, vitamins, and a carbon source like acetate or glucose.

3. Difference between Macronutrient and Micronutrient:

   • Macronutrients are required in large quantities by organisms and include elements such as carbon, nitrogen, and phosphorus. Micronutrients are required in smaller quantities and include elements like iron, zinc, and manganese.

4. Why Chlamydomonas and humans need phosphate (PO4) and iron (Fe):

   • Phosphate is essential for DNA, RNA, and ATP synthesis, while iron is required for chlorophyll and hemoglobin synthesis, electron transport, and enzyme function.

5. Growth and doubling time:

   • Chlamydomonas grows exponentially, doubling in population size in approximately 10 hours at 25°C. This means that the population doubles every 10 hours under optimal growth conditions.

6. Microbial growth curve:

   • The growth curve shows three phases: lag phase (adaptation to new environment), exponential phase (rapid growth), and stationary phase (nutrient depletion and waste accumulation).

7. Chlamydomonas phylogeny and relationship to plants and animals:

   • Chlamydomonas is a green alga and is evolutionarily related to land plants. It shares a common ancestor with plants but is more closely related to animals than to plants.

8. Explanation for why Chlamydomonas and humans have flagella but plants do not:

   • The simplest explanation is that Chlamydomonas and humans are both motile organisms that require flagella for movement, while plants are typically non-motile and do not require flagella for locomotion.

9. Basics of cilia structure and ciliopathies:

   • Cilia are composed of microtubules arranged in a 9+2 pattern and are involved in cell motility and signaling. Mutations in cilia-related proteins can cause ciliopathies, which are associated with various human diseases.

10. Distinctions between motile and non-motile flagella:

    • Motile flagella, like those in Chlamydomonas, are involved in movement, while non-motile flagella, like those in sensory cells, are involved in sensory reception.

Mental Floss Questions:

1. Characteristics of Chlamy that make it a "model experimental system":

   • Chlamydomonas is unicellular, haploid, photosynthetic, and genetically tractable, making it an ideal model organism for studying various biological processes.

2. Reason Chlamy stops dividing during stationary phase:

   • Chlamy stops dividing during stationary phase due to nutrient depletion, accumulation of waste products, and changes in environmental conditions.

3. Analysis of 7,476 Chlamy proteins:

   • Proteins common to only Chlamy and humans, only Chlamy and Arabidopsis, and all three species likely reflect shared biological functions and evolutionary relationships between these organisms.

From Textbook Readings:

1. Meaning of potential, kinetic, and chemical energy:

   • Potential energy: Energy stored in an object due to its position or state. Examples include gravitational potential energy and chemical energy stored in bonds.

   • Kinetic energy: Energy possessed by an object in motion. Examples include the movement of molecules in a gas and the motion of a rolling ball.

   • Chemical energy: Energy stored in the bonds of chemical compounds. Examples include the energy stored in glucose molecules and the energy released during combustion reactions.

2. Distinction between Closed, Open, and Isolated systems:

   • Closed system: A system where energy can exchange with its surroundings but not matter. Example: A sealed container.

   • Open system: A system where both energy and matter can exchange with its surroundings. Example: An organism exchanging gases and nutrients with its environment.

   • Isolated system: A system that cannot exchange energy or matter with its surroundings. Example: The universe as a whole.

3. First Law of Thermodynamics:

   • Energy cannot be created or destroyed; it can only change forms. Example: The energy from food is converted into mechanical energy by muscles.

4. Second Law of Thermodynamics:

   • The total entropy of an isolated system always increases over time. Example: Heat spontaneously flows from hot objects to cold objects.

5. Four levels of protein structure:

   • Primary structure: Sequence of amino acids in a polypeptide chain, held together by peptide bonds.

   • Secondary structure: Regular repeating patterns such as alpha helices and beta sheets, stabilized by hydrogen bonds.

   • Tertiary structure: Three-dimensional folding of the polypeptide chain, stabilized by various interactions including hydrogen bonds, disulfide bonds, hydrophobic interactions, and electrostatic interactions.

   • Quaternary structure: Arrangement of multiple polypeptide subunits, stabilized by the same interactions as tertiary structure.

From Class:

1. Why we need to eat:

   • We need to eat to obtain nutrients and energy necessary for cellular processes, growth, and maintenance of bodily functions.

2. Flow of energy through the biosphere and concept of carbon compounds being reduced or oxidized:

   • Carbon compounds can be reduced or oxidized during metabolic processes. Autotrophs reduce carbon compounds, while heterotrophs oxidize them to obtain energy.

3. Work and breakdown in living systems and application of the second law of thermodynamics:

   • Living systems perform work and maintain low entropy by continuously importing energy from the environment. The second law of thermodynamics applies to living systems as entropy represents energy spreading or disorder.

4. Definition of free energy (ΔG), spontaneous reactions, enthalpy, and entropy:

   • Free energy (ΔG) is the energy available to do work in a system. Spontaneous reactions have a negative ΔG. Enthalpy (H) is the total energy of a system, while entropy (S) is a measure of the disorder or randomness in a system.

5. Distinction among exothermic, endothermic, exergonic, and endergonic reactions:

   • Exothermic reactions release energy to the surroundings, while endothermic reactions absorb energy from the surroundings. Exergonic reactions release free energy, while endergonic reactions require energy input.

6. Role of enzymes in increasing the rate of spontaneous and non-spontaneous reactions:

   • Enzymes lower the activation energy barrier for both spontaneous and non-spontaneous reactions, allowing them to occur more readily.

7. Features of the exergonic reaction energy profile and how enzymes affect it:

   • The exergonic reaction energy profile includes the ΔG, transition state, and activation energy. Enzymes stabilize the transition state, lowering the activation energy required for the reaction.

8. Importance of enzymes in the evolution of life:

   • Enzymes catalyze biochemical reactions, allowing organisms to perform essential functions more efficiently. Their presence likely contributed to the evolution of complex life forms.

9. Basics of protein folding and properties of urea that denature proteins:

   • Correct protein folding requires the amino acid sequence, appropriate environmental conditions, and the absence of denaturants like urea, which disrupt hydrogen bonding and hydrophobic interactions.

10. Free energy and the Energy funneling model of protein folding:

    • The energy funneling model suggests that proteins fold by progressively lowering their free energy, leading to a stable native structure. Chaperones assist in this process.

11. Basics of enzyme structure and the catalytic cycle:

    • Enzymes typically have an active site where substrates bind and undergo a catalytic cycle involving substrate binding, catalysis, and product release.

From Homework:

1. Processes explaining the shape of a Growth vs Temperature curve in E. coli:

   • The shape of the curve reflects the optimal temperature for growth, as well as the effects of temperature on enzyme activity

From Textbook Readings:

1. Meaning of potential, kinetic, and chemical energy:

   • Potential energy: Energy stored in an object due to its position or state. Examples include gravitational potential energy and chemical energy stored in bonds.

   • Kinetic energy: Energy possessed by an object in motion. Examples include the movement of molecules in a gas and the motion of a rolling ball.

   • Chemical energy: Energy stored in the bonds of chemical compounds. Examples include the energy stored in glucose molecules and the energy released during combustion reactions.

2. Distinction between Closed, Open, and Isolated systems:

   • Closed system: A system where energy can exchange with its surroundings but not matter. Example: A sealed container.

   • Open system: A system where both energy and matter can exchange with its surroundings. Example: An organism exchanging gases and nutrients with its environment.

   • Isolated system: A system that cannot exchange energy or matter with its surroundings. Example: The universe as a whole.

3. First Law of Thermodynamics:

   • Energy cannot be created or destroyed; it can only change forms. Example: The energy from food is converted into mechanical energy by muscles.

4. Second Law of Thermodynamics:

   • The total entropy of an isolated system always increases over time. Example: Heat spontaneously flows from hot objects to cold objects.

5. Four levels of protein structure:

   • Primary structure: Sequence of amino acids in a polypeptide chain, held together by peptide bonds.

   • Secondary structure: Regular repeating patterns such as alpha helices and beta sheets, stabilized by hydrogen bonds.

   • Tertiary structure: Three-dimensional folding of the polypeptide chain, stabilized by various interactions including hydrogen bonds, disulfide bonds, hydrophobic interactions, and electrostatic interactions.

   • Quaternary structure: Arrangement of multiple polypeptide subunits, stabilized by the same interactions as tertiary structure.

From Class:

1. Why we need to eat:

   • We need to eat to obtain nutrients and energy necessary for cellular processes, growth, and maintenance of bodily functions.

2. Flow of energy through the biosphere and concept of carbon compounds being reduced or oxidized:

   • Carbon compounds can be reduced or oxidized during metabolic processes. Autotrophs reduce carbon compounds, while heterotrophs oxidize them to obtain energy.

3. Work and breakdown in living systems and application of the second law of thermodynamics:

   • Living systems perform work and maintain low entropy by continuously importing energy from the environment. The second law of thermodynamics applies to living systems as entropy represents energy spreading or disorder.

4. Definition of free energy (ΔG), spontaneous reactions, enthalpy, and entropy:

   • Free energy (ΔG) is the energy available to do work in a system. Spontaneous reactions have a negative ΔG. Enthalpy (H) is the total energy of a system, while entropy (S) is a measure of the disorder or randomness in a system.

5. Distinction among exothermic, endothermic, exergonic, and endergonic reactions:

   • Exothermic reactions release energy to the surroundings, while endothermic reactions absorb energy from the surroundings. Exergonic reactions release free energy, while endergonic reactions require energy input.

6. Role of enzymes in increasing the rate of spontaneous and non-spontaneous reactions:

   • Enzymes lower the activation energy barrier for both spontaneous and non-spontaneous reactions, allowing them to occur more readily.

7. Features of the exergonic reaction energy profile and how enzymes affect it:

   • The exergonic reaction energy profile includes the ΔG, transition state, and activation energy. Enzymes stabilize the transition state, lowering the activation energy required for the reaction.

8. Importance of enzymes in the evolution of life:

   • Enzymes catalyze biochemical reactions, allowing organisms to perform essential functions more efficiently. Their presence likely contributed to the evolution of complex life forms.

9. Basics of protein folding and properties of urea that denature proteins:

   • Correct protein folding requires the amino acid sequence, appropriate environmental conditions, and the absence of denaturants like urea, which disrupt hydrogen bonding and hydrophobic interactions.

10. Free energy and the Energy funneling model of protein folding:

    • The energy funneling model suggests that proteins fold by progressively lowering their free energy, leading to a stable native structure. Chaperones assist in this process.

11. Basics of enzyme structure and the catalytic cycle:

    • Enzymes typically have an active site where substrates bind and undergo a catalytic cycle involving substrate binding, catalysis, and product release.

From Homework:

1. Processes explaining the shape of a Growth vs Temperature curve in E. coli:

   • The shape of the curve reflects the optimal temperature for growth, as well as the effects of temperature on enzyme activity

From Textbook Reading:

1. Two phases of photosynthesis:

   • Light reactions: Convert light energy into chemical energy in the form of ATP and NADPH.

   • Calvin cycle: Uses ATP and NADPH produced in the light reactions to fix carbon dioxide and synthesize carbohydrates.

2. Structure and function of a photosystem:

   • A photosystem consists of antenna pigments (such as chlorophyll and carotenoids) that capture light energy and transfer it to a reaction center chlorophyll. The reaction center chlorophyll initiates the light-dependent reactions by transferring excited electrons to an electron acceptor.

From Class Video:

1. Global primary productivity and why there's little photosynthesis in oceans:

   • Factors such as nutrient availability, light availability, and temperature limit primary productivity in oceans. Nutrient limitation, particularly of nitrogen and phosphorus, can significantly restrict photosynthesis in marine environments.

2. Definitions:

   • Photosynthesis: The process by which green plants and some other organisms use sunlight to synthesize food from carbon dioxide and water.

   • Endergonic: A reaction that requires energy input to proceed.

   • Redox reaction: A chemical reaction in which one substance is oxidized (loses electrons) and another is reduced (gains electrons).

3. Extremophile mental floss:

   • Differences in growth vs temperature curves and adaptations of enzymes like hexokinase to different habitats.

4. Bacteriorhodopsin:

   • Bacteriorhodopsin is a light-driven proton pump found in archaea. It captures light energy to transport protons across the membrane, generating a proton gradient.

5. Structural features of the chloroplast:

   • Chloroplasts have an outer membrane, inner membrane, stroma, thylakoid membrane system, and grana. Light reactions occur in the thylakoid membranes, while the Calvin cycle takes place in the stroma.

6. Electron transport chain and ATP synthase:

   • The electron transport chain transports electrons from photosystem II to photosystem I, generating a proton gradient across the thylakoid membrane. ATP synthase uses the energy from the proton gradient to produce ATP.

7. Redox potential and electron flow:

   • Redox potential determines the tendency of a molecule to gain or lose electrons. In photosynthesis, chlorophyll's redox potential changes upon photon absorption, driving electron flow through the photosystems.

8. Processes involving P680, P680*, and P680+:

   • P680 is the reaction center chlorophyll in photosystem II. P680* is the excited state of P680, which donates electrons to the electron transport chain. P680+ is the oxidized form of P680.

9. Why Chlamy is sometimes negatively phototactic:

   • Chlamydomonas exhibits negative phototaxis under certain conditions, likely to avoid excessive light exposure and potential damage to photosystems.

10. Role of chloroplast protein synthesis in PSII repair:

    • Chloroplast protein synthesis is involved in repairing damaged PSII complexes, particularly the D1 protein.

11. Calvin cycle:

    • The Calvin cycle takes place in the stroma and uses ATP and NADPH produced in the light reactions to fix carbon dioxide and produce sugars. It consists of three phases: carbon fixation, reduction, and regeneration.

12. Basic differences between anoxygenic and oxygenic photosynthesis:

    • Anoxygenic photosynthesis occurs in bacteria and does not produce oxygen as a byproduct, whereas oxygenic photosynthesis, found in plants and algae, releases oxygen.

13. Evolutionary advantage of oxygenic photosynthesis:

    • Oxygenic photosynthesis produces oxygen as a byproduct, leading to the oxygenation of the atmosphere, which enabled the evolution of aerobic organisms and more efficient energy metabolism.

From Textbook Reading:

1. Two phases of photosynthesis:

   • Light reactions: Convert light energy into chemical energy in the form of ATP and NADPH.

   • Calvin cycle: Uses ATP and NADPH produced in the light reactions to fix carbon dioxide and synthesize carbohydrates.

2. Structure and function of a photosystem:

   • A photosystem consists of antenna pigments (such as chlorophyll and carotenoids) that capture light energy and transfer it to a reaction center chlorophyll. The reaction center chlorophyll initiates the light-dependent reactions by transferring excited electrons to an electron acceptor.

From Class Video:

1. Global primary productivity and why there's little photosynthesis in oceans:

   • Factors such as nutrient availability, light availability, and temperature limit primary productivity in oceans. Nutrient limitation, particularly of nitrogen and phosphorus, can significantly restrict photosynthesis in marine environments.

2. Definitions:

   • Photosynthesis: The process by which green plants and some other organisms use sunlight to synthesize food from carbon dioxide and water.

   • Endergonic: A reaction that requires energy input to proceed.

   • Redox reaction: A chemical reaction in which one substance is oxidized (loses electrons) and another is reduced (gains electrons).

3. Extremophile mental floss:

   • Differences in growth vs temperature curves and adaptations of enzymes like hexokinase to different habitats.

4. Bacteriorhodopsin:

   • Bacteriorhodopsin is a light-driven proton pump found in archaea. It captures light energy to transport protons across the membrane, generating a proton gradient.

5. Structural features of the chloroplast:

   • Chloroplasts have an outer membrane, inner membrane, stroma, thylakoid membrane system, and grana. Light reactions occur in the thylakoid membranes, while the Calvin cycle takes place in the stroma.

6. Electron transport chain and ATP synthase:

   • The electron transport chain transports electrons from photosystem II to photosystem I, generating a proton gradient across the thylakoid membrane. ATP synthase uses the energy from the proton gradient to produce ATP.

7. Redox potential and electron flow:

   • Redox potential determines the tendency of a molecule to gain or lose electrons. In photosynthesis, chlorophyll's redox potential changes upon photon absorption, driving electron flow through the photosystems.

8. Processes involving P680, P680*, and P680+:

   • P680 is the reaction center chlorophyll in photosystem II. P680* is the excited state of P680, which donates electrons to the electron transport chain. P680+ is the oxidized form of P680.

9. Why Chlamy is sometimes negatively phototactic:

   • Chlamydomonas exhibits negative phototaxis under certain conditions, likely to avoid excessive light exposure and potential damage to photosystems.

10. Role of chloroplast protein synthesis in PSII repair:

    • Chloroplast protein synthesis is involved in repairing damaged PSII complexes, particularly the D1 protein.

11. Calvin cycle:

    • The Calvin cycle takes place in the stroma and uses ATP and NADPH produced in the light reactions to fix carbon dioxide and produce sugars. It consists of three phases: carbon fixation, reduction, and regeneration.

12. Basic differences between anoxygenic and oxygenic photosynthesis:

    • Anoxygenic photosynthesis occurs in bacteria and does not produce oxygen as a byproduct, whereas oxygenic photosynthesis, found in plants and algae, releases oxygen.

13. Evolutionary advantage of oxygenic photosynthesis:

    • Oxygenic photosynthesis produces oxygen as a byproduct, leading to the oxygenation of the atmosphere, which enabled the evolution of aerobic organisms and more efficient energy metabolism.

Basics of Measuring Respiration in Isolated Mitochondria:

1. Materials Needed:

   • Isolated mitochondria

   • Respiration chamber with electrodes for measuring oxygen consumption

   • Substrates and cofactors (e.g., NADH, ADP, Pi)

   • Uncoupler (e.g., FCCP)

   • Buffer solution

2. Procedure:

   • Isolated mitochondria are placed in the respiration chamber containing a buffer solution.

   • Baseline oxygen consumption is measured.

   • Various substrates and cofactors are added sequentially, such as NADH, ADP, and Pi.

   • Oxygen consumption is measured after each addition.

   • Uncoupler is added to assess the maximal respiratory capacity.

Change in Respiration Rate with Each Addition:

1. NADH Addition:

   • NADH provides electrons to the electron transport chain, stimulating oxygen consumption as electrons are transferred through the respiratory complexes.

2. ADP & Pi Addition:

   • ADP and Pi stimulate oxygen consumption by promoting ATP synthesis. The increase in ADP levels activates the electron transport chain to drive ATP synthesis via oxidative phosphorylation.

3. Uncoupler Addition:

   • Uncouplers like FCCP disrupt the proton gradient across the inner mitochondrial membrane, uncoupling electron transport from ATP synthesis. Oxygen consumption increases drastically as mitochondria work to maintain the proton gradient.

Respiratory Control:

• Respiratory control refers to the regulation of mitochondrial respiration by the availability of ADP and the demand for ATP. It is mediated by the coupling of electron transport to ATP synthesis via oxidative phosphorylation. When ADP levels increase (indicating low ATP), respiration increases to produce more ATP.

Autotrophic Metabolism:

• Autotrophic metabolism involves the ability of organisms to synthesize their own organic molecules from inorganic sources. In plants, chloroplasts carry out photosynthesis to produce sugars, which are then used in cellular respiration to generate ATP.

Roles of G3P Exported from the Chloroplast:

1. Energy Source:

   • G3P (glyceraldehyde-3-phosphate) can be used as an energy source in cellular respiration by entering the glycolytic pathway to produce ATP.

2. Carbon Source:

   • G3P exported from the chloroplast can serve as a carbon source for biosynthetic pathways in the cytosol, contributing to the synthesis of other organic molecules.

Can Chlamy Grow in the Dark? Why Acetate and Not Glucose?

• Chlamydomonas can grow in the dark using acetate as a carbon source because it can perform mixotrophic metabolism. Acetate can be metabolized via the glyoxylate cycle to produce intermediates for biosynthesis. Chlamydomonas prefers acetate over glucose because it can utilize acetate more efficiently in the absence of light.

Mixotrophic Metabolism:

• Mixotrophic metabolism refers to the ability of an organism to use both organic and inorganic sources of carbon and energy for growth. In Chlamydomonas, mixotrophic metabolism allows it to grow in the presence of light using photosynthesis and in the dark using organic carbon sources like acetate.

Gas Exchange in Respiration and Photosynthesis:

• Respiration:

  • Consumes O2 and produces CO2 as a byproduct.

• Photosynthesis:

  • Consumes CO2 and produces O2 as a byproduct.

Underestimation of Rates in Whole Chlamy Cells:

• Measurements of O2 production or CO2 fixation in whole Chlamy cells underestimate the actual rate due to concurrent respiration, which consumes O2 and produces CO2.

Measuring Photosynthesis Using Gas Exchange:

• Gas exchange can be measured by monitoring changes in O2 and CO2 concentrations over time. The rate of CO2 fixation can be quantified in terms of carbon fixation units (e.g., µmol CO2 fixed per unit time).

Light Response Curve:

• Linear Portion (Light-limited Region):

  • In this region, photosynthesis is limited by light availability. Increasing light intensity leads to an increase in photosynthetic rate.

• Plateau (Light-saturated Region):

  • In this region, photosynthesis becomes saturated, and further increases in light intensity do not significantly increase the photosynthetic rate.

Increasing Light-Saturated Rate:

• The light-saturated rate can be increased by increasing the content of Rubisco or the availability of CO2.

Determining Rate of Cellular Respiration:

• The rate of cellular respiration can be determined by measuring O2 consumption or CO2 production in the presence of an appropriate substrate.

Light Compensation Point:

• The light compensation point is the light intensity at which the rate of photosynthesis equals the rate of respiration. Below this point, plants lose carbon due to respiration, and above this point, they gain carbon through photosynthesis.

Rate of Reaction vs. Enzyme Concentration Curve:

• Initially, the rate of reaction increases linearly with increasing enzyme concentration. However, at high enzyme concentrations, the rate plateaus as all substrate molecules become saturated with enzymes.

Adaptation to Low Temperature:

• One adaptation to low temperature is to increase enzyme concentration to compensate for decreased enzyme activity at lower temperatures.

Rate of Reaction vs. Substrate Concentration Curve:

• Initially, the rate of reaction increases with increasing substrate concentration until it reaches a maximum (Vmax), where all enzyme active sites are saturated with substrate.

Parameters Vmax and Km:

• Vmax: Maximum velocity of an enzyme-catalyzed reaction when the enzyme is fully saturated with substrate.

• Km: Substrate concentration at which the reaction rate is half of Vmax.

Competitive Inhibitor:

• Competitive inhibitors compete with substrate for binding to the active site of an enzyme. They increase Km but do not affect Vmax.

Non-competitive (Allosteric) Regulation:

• Non-competitive inhibitors bind to the enzyme at a site other than the active site, causing a conformational change that decreases enzyme activity. They do not affect Km but reduce Vmax.

Allosteric Regulation:

• Allosteric activators bind to the enzyme and increase its activity, while allosteric inhibitors decrease enzyme activity.

Regulation of Enzyme Activity:

• Regulating enzyme activity at the level of enzyme activity allows for rapid responses to changing cellular conditions compared to transcriptional or translational regulation.

Major Metabolites and Enzyme Regulation:

• Switch ON: ATP, NADPH

• Switch OFF: ADP, NADP+, NADH

Three Domains of Life:

1. Bacteria:

   • Prokaryotic cells with peptidoglycan cell walls.

   • Unicellular organisms with various shapes.

   • No membrane-bound organelles.

2. Archaea:

   • Prokaryotic cells with unique cell membrane compositions.

   • Often found in extreme environments.

   • Some share characteristics with both bacteria and eukaryotes.

3. Eukarya:

   • Eukaryotic cells with membrane-bound organelles and a nucleus.

   • Includes plants, animals, fungi, and protists.

   • Typically larger and more complex than prokaryotes.

Role of rRNA Gene Sequencing:

• rRNA gene sequencing is used to determine the evolutionary relationships between organisms. It provides insights into their genetic relatedness and helps in reconstructing phylogenetic trees.

LUCA (Last Universal Common Ancestor):

• LUCA is the hypothetical organism from which all life on Earth descended.

• Common characteristics found across all domains include the use of DNA as genetic material, protein synthesis via ribosomes, and a similar genetic code.

Evolution of Life:

• Life evolved from simple anaerobic organisms to more complex aerobic organisms.

• Anaerobic metabolism preceded aerobic metabolism, with the development of oxygenic photosynthesis leading to the accumulation of oxygen in the atmosphere.

Complexity of Eukaryotes:

• Eukaryotes are more complex than prokaryotes due to the presence of membrane-bound organelles, including mitochondria and chloroplasts.

• Mitochondria evolved from aerobic bacteria, providing eukaryotes with increased energy efficiency.

Great Oxygenation Event:

• The Great Oxygenation Event refers to the rise of oxygen in the Earth's atmosphere around 2.4 billion years ago.

• Increased oxygen levels allowed for the evolution of more complex organisms with multiple cell types, larger genomes, and increased metabolic capabilities.

Genome Size in Prokaryotes:

• Prokaryotes have relatively small genomes compared to eukaryotes due to their simpler cellular organization and lack of introns and non-coding regions.

Origin of Endomembrane System and Nuclear Membrane:

• The endomembrane system and nuclear membrane likely originated from invaginations of the plasma membrane in ancestral eukaryotes.

Endosymbiosis:

• Endosymbiosis is the theory that mitochondria and chloroplasts originated from engulfed prokaryotic cells that formed a symbiotic relationship with the host cell.

• Evidence includes the presence of their own DNA, similar to bacterial DNA, and the presence of double membranes.

Horizontal Gene Transfer (HGT):

• HGT is the transfer of genetic material between organisms that are not directly related.

• HGT between mitochondria/chloroplasts and the nucleus is believed to occur, leading to gene transfer from organelles to the nucleus.

Signal Peptide and Protein Targeting:

• Signal peptides target nuclear-encoded proteins to organelles like mitochondria and chloroplasts.

• These peptides are recognized by specific receptors on the organelle membranes, leading to import of the protein.

Three Domains vs Two Domain Tree of Life:

• The three-domain tree of life separates bacteria, archaea, and eukaryotes into distinct domains based on genetic and biochemical differences.

• Eukaryotic signature proteins and the discovery of Asgard archaea have challenged the traditional two-domain tree.

Eukaryotic Cell as a Chimera:

• The eukaryotic cell is considered a chimera because it contains genetic material and cellular components from both bacteria (e.g., mitochondria) and archaea (e.g., some genetic machinery).

Origin of Membrane Lipids in Eukaryotes:

• The surprising thing about the origin of membrane lipids in eukaryotes is that they are believed to have originated from archaea, rather than bacteria.

Three Genomes in Chlamy:

• Chlamydomonas has three genomes: nuclear, mitochondrial, and chloroplast.

• This means that each compartment requires its own transcriptional and translational machinery for protein synthesis.

Antibiotic:

An antibiotic is a type of antimicrobial substance produced naturally by microorganisms or synthesized in the laboratory. It is used to inhibit the growth of or kill bacteria.

Major Aspects of Bacterial Cell Structure:

• Cell Wall: Provides structural support and protection. It's present in both Gram-positive and Gram-negative bacteria.

• Plasmid: Circular DNA molecules that can replicate independently of the bacterial chromosome. They often carry genes for antibiotic resistance.

Structural Differences in Cell Surface:

• Gram-positive bacteria: Thick peptidoglycan layer, no outer membrane.

• Gram-negative bacteria: Thin peptidoglycan layer, outer membrane containing lipopolysaccharides (LPS).

Peptidoglycan Cell Wall:

• Peptidoglycan is a mesh-like structure composed of alternating sugar molecules and short peptides.

• Transpeptidase enzymes cross-link the peptide chains, providing strength to the cell wall.

The action of Penicillin:

• Penicillin inhibits the action of transpeptidase enzymes, preventing cross-linking of peptidoglycan during cell wall synthesis.

• This weakens the cell wall, leading to cell lysis and bacterial death.

Mode of Action of Major Antibiotics:

• Inhibition of Cell Wall Synthesis: Penicillins, cephalosporins.

• Inhibition of Protein Synthesis: Tetracyclines, macrolides.

• Disruption of Cell Membrane: Polymyxins.

• Inhibition of Nucleic Acid Synthesis: Fluoroquinolones, rifampin.

Why Antibiotics Don't Kill Us:

• Antibiotics target bacterial processes or structures that are absent or significantly different in human cells.

• For example, human cells lack a cell wall like bacteria, so antibiotics targeting cell wall synthesis are specific to bacteria.

Mitochondria Are Prokaryotic:

• Mitochondria are believed to have originated from an endosymbiotic event where a prokaryotic cell was engulfed by another cell.

• They retain some characteristics of their prokaryotic ancestors, such as their own DNA and ribosomes.

Assay to Detect Antibiotic-Resistant Bacteria:

• Antibiotic susceptibility testing involves culturing bacteria in the presence of antibiotics and observing their growth. The disk diffusion method and broth dilution method are common assays.

Two Ways Antibiotic Resistance Is Acquired:

1. Horizontal Gene Transfer: Transfer of resistance genes between bacteria through conjugation, transformation, or transduction.

2. Random Mutation: Changes in bacterial DNA that confer resistance to antibiotics.

Random Mutation:

• Random mutation refers to spontaneous changes in the DNA sequence of an organism's genome. These mutations can occur during DNA replication and can be beneficial, harmful, or neutral.

Bacterial Conjugation:

• Bacterial conjugation is a mechanism of horizontal gene transfer where genetic material is transferred from a donor bacterium to a recipient bacterium through direct cell-to-cell contact via a conjugation pilus.

Major Mechanisms of Resistance:

1. Efflux Pump: Proteins that actively pump antibiotics out of the bacterial cell.

2. Downregulation of Porin: Reduction in the expression of porin proteins, which decreases the entry of antibiotics into the cell.

Role of Loss of Porin in Acquisition of Resistance in Klebsiella:

• Loss of porin proteins in Klebsiella pneumoniae reduces the permeability of the outer membrane, making it more difficult for antibiotics to enter the cell, thus conferring resistance.

Comparison of Prokaryotic and Eukaryotic Transcription and Translation:

Complementary Base Pairing:

• Adenine (A) - Thymine (T)

• Cytosine (C) - Guanine (G)

• Adenine (A) - Uracil (U) (in RNA)

• Guanine (G) - Cytosine (C)

Why Transcription and Translation Can Occur Simultaneously in Prokaryotic Cells but Not in Eukaryotic Cells:

• In prokaryotic cells, transcription and translation occur in the same compartment (cytoplasm), allowing them to happen simultaneously. Additionally, prokaryotic mRNA is typically translated as it is being transcribed.

• In eukaryotic cells, transcription occurs in the nucleus, while translation occurs in the cytoplasm. The mRNA transcript must first be processed and transported out of the nucleus before translation can begin, making simultaneous transcription and translation impossible.

Central Dogma and Its Inaccuracy:

• The central dogma states that DNA is transcribed into RNA, which is then translated into protein.

• It's not entirely correct because many RNA molecules do not serve as templates for protein synthesis; they have various other functions such as regulatory roles, catalysis, or structural support.

Structure of Ribosomes:

• Ribosomes consist of ribosomal RNA (rRNA) and proteins.

• rRNA plays a catalytic role in peptide bond formation during translation, while proteins provide structural support.

tRNA:

• Transfer RNA (tRNA) molecules carry specific amino acids to the ribosome during translation.

• Each tRNA has an anticodon that pairs with the codon on the mRNA.

Basic Steps in Translation:

1. Initiation: mRNA binds to the small ribosomal subunit, and the initiator tRNA binds to the start codon.

2. Elongation: Amino acids are added to the growing polypeptide chain according to the mRNA codons.

3. Termination: Translation stops when a stop codon is reached, and the completed polypeptide is released from the ribosome.

Protein Coding Gene vs. Non-Protein Coding Gene:

• Protein Coding Gene: Contains exons that code for proteins and introns that are spliced out during mRNA processing.

• Non-Protein Coding Gene: May produce functional RNA molecules such as rRNA, tRNA, microRNAs, or regulatory RNAs.

Polyribosomes:

• Polyribosomes (or polysomes) are clusters of ribosomes translating a single mRNA molecule simultaneously.

• They appear as "beads on a string" in electron micrographs.

Regulatory Sequences of Transcription:

• In prokaryotes, regulatory sequences include promoter regions, operators, and transcription factors.

• In eukaryotes, regulatory sequences include promoter regions, enhancers, silencers, and transcription factors.

Mechanism of Intron Splicing:

• Introns are removed from pre-mRNA by a process called splicing, mediated by the spliceosome.

• The spliceosome recognizes specific sequences at the intron-exon boundaries and cuts the intron, allowing exons to be joined together.

Alternative Splicing:

• Alternative splicing is a process where different combinations of exons are spliced together, resulting in multiple mRNA isoforms from a single gene.

• It can increase protein diversity but can also lead to disease if deregulated.

Cell Compartments and Processes:

Differential vs Temporal Gene Expression:

• Differential Gene Expression: Refers to the differences in the expression levels of genes between different cell types or conditions.

• Temporal Gene Expression: Refers to changes in gene expression over time, such as during development or in response to environmental cues.

Levels of Regulation:

• Transcriptional Regulation: Control of gene expression at the level of transcription initiation by transcription factors and chromatin remodeling.

• Posttranscriptional Regulation: Regulation of mRNA processing, stability, and transport, including splicing and RNA editing.

• Translational Regulation: Control of mRNA translation efficiency by regulatory proteins and non-coding RNAs.

• Posttranslational Regulation: Regulation of protein activity, stability, and localization through processes such as phosphorylation, ubiquitination, and proteolysis.

Lac Operon:

• The lac operon in E. coli consists of genes lacZ, lacY, and lacA, involved in lactose metabolism.

• LacZ encodes β-galactosidase, which converts lactose to glucose and galactose.

• LacY encodes lactose permease, which transports lactose into the cell.

• LacA encodes thiogalactoside transacetylase, which is involved in lactose metabolism.

Mechanism of Pre-mRNA Splicing:

• Pre-mRNA splicing removes introns and joins exons to produce mature mRNA.

• It is catalyzed by the spliceosome, which recognizes the 5' splice site, branch point sequence, and 3' splice site.

• Mutations in splice sites or splicing factors can lead to aberrant mRNA and protein production.

Beta-Thalassemia and RNA Editing:

• Beta-thalassemia is caused by mutations in the beta-globin gene, leading to abnormal hemoglobin production.

• In RNA editing, ApoB100 is edited to produce ApoB48 by a deamination reaction mediated by the enzyme APOBEC1.

• Mutations in the intron outside the splice sites can affect splicing and cause beta-thalassemia.

Telomerase and Cell Senescence:

• Telomerase is an enzyme that adds telomere repeats to chromosome ends, preventing their shortening during cell division.

• It is activated in cancer cells to maintain telomere length and in stem cells for self-renewal.

• The Hayflick limit refers to the maximum number of cell divisions a cell undergoes before entering senescence.

Stem Cells:

• Stem cells are undifferentiated cells capable of self-renewal and differentiation into various cell types.

• Adult stem cells are multipotent and found in tissues like bone marrow.

• Stem cells divide asymmetrically or symmetrically to self-renew or differentiate into progenitor cells.

• Epidermal stem cells maintain skin homeostasis, while intestinal stem cells regenerate the intestinal epithelium.

Induced Pluripotent Stem (iPS) Cells:

• iPS cells are generated by reprogramming somatic cells to an embryonic-like state using transcription factors.

• They have potential applications in regenerative medicine, disease modeling, and drug discovery.

• iPS cells have been used to treat macular degeneration, burns, and myocardial infarction, demonstrating their therapeutic potential.

Molecular Mechanisms Underlying Alzheimer's Disease (AD):

1. Structure of Neurons and Synaptic Communication:

   • Neurons are specialized cells in the nervous system responsible for transmitting information.

   • They communicate at the synaptic cleft through neurotransmitters released by the presynaptic neuron and received by the postsynaptic neuron.

2. Cholinergic Neuron Downregulation:

   • Cholinergic neurons, which produce the neurotransmitter acetylcholine, are downregulated in AD.

   • This downregulation contributes to cognitive decline and memory impairment in AD patients.

3. Beta-Amyloid Plaque Formation:

   • Beta-amyloid plaques are formed by the aggregation of amyloid-beta peptides, derived from the cleavage of the amyloid precursor protein (APP).

   • Aggregated beta-amyloid peptides accumulate outside neurons, disrupting neuronal function and leading to neurotoxicity.

4. Neurofibrillary Tangle Formation:

   • Neurofibrillary tangles are formed by abnormal phosphorylation of tau proteins, leading to their aggregation into insoluble filaments.

   • These tangles disrupt the microtubule structure within neurons, impairing neuronal transport and causing cell death.

5. Common Drugs for AD and Their Targets:

   • Cholinesterase Inhibitors (e.g., Donepezil, Rivastigmine): Inhibit the breakdown of acetylcholine, increasing its levels in the brain.

   • NMDA Receptor Antagonists (e.g., Memantine): Blocks excessive activation of glutamate receptors, preventing excitotoxicity.

6. Genetic Risk Factors of AD:

   • Mutations in genes such as APP, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) increase the risk of developing familial AD.

   • Apolipoprotein E (APOE) genotype, particularly APOE ε4 allele, is a major genetic risk factor for sporadic late-onset AD.

7. AD Drug Development:

   • There are ongoing efforts to develop new drugs for AD targeting various aspects of the disease pathology, including beta-amyloid aggregation, tau pathology, neuroinflammation, and synaptic dysfunction.

   • Some potential drugs in the developmental pipeline include monoclonal antibodies targeting beta-amyloid, tau aggregation inhibitors, and neuroprotective agents.

Summary:

Alzheimer's Disease involves complex molecular mechanisms including the formation of beta-amyloid plaques and neurofibrillary tangles, downregulation of cholinergic neurons, and genetic factors. Current treatments aim to alleviate symptoms by targeting neurotransmitter systems and neuroprotective mechanisms, but ongoing research is focused on developing disease-modifying therapies to slow or halt disease progression.

Genetic Heterogeneity of Cancers:

1. Inter-tumoral Heterogeneity vs. Intra-tumoral Heterogeneity:

   • Inter-tumoral Heterogeneity: Refers to differences between tumors of different patients, such as different genetic mutations, gene expression profiles, and clinical behaviors.

   • Intra-tumoral Heterogeneity: Refers to genetic and phenotypic differences within a single tumor mass, arising from genetic mutations, epigenetic changes, and microenvironmental factors.

2. Clonal Evolution of Cancer:

   • Cancer cells undergo clonal evolution, where they accumulate genetic mutations and evolve into subpopulations with diverse phenotypes.

   • Selective pressures such as therapy, immune response, and microenvironmental conditions drive the expansion of certain clones with survival advantages.

3. Drug Resistance and Relapse:

   • Cancer cells can develop resistance to drug treatment through various mechanisms, including mutations in drug targets, activation of alternative signaling pathways, and upregulation of drug efflux pumps.

   • This leads to treatment failure and relapse, as resistant clones survive and proliferate, driving disease progression.

4. Driver Mutations vs. Passenger Mutations:

   • Driver Mutations: Genetic alterations that directly contribute to cancer development by conferring a growth advantage to the affected cells.

   • Passenger Mutations: Genetic alterations that do not confer a selective advantage but accumulate during clonal evolution.

5. DNA Damage and Mutator Phenotype:

   • Endogenous factors (e.g., reactive oxygen species) and exogenous exposures (e.g., UV radiation, carcinogens) can induce DNA damage, leading to mutations and genomic instability.

   • Persistent DNA damage and impaired DNA repair mechanisms can result in a mutator phenotype, contributing to cancer development and chemotherapy resistance.

6. Proto-oncogenes vs. Tumor Suppressor Genes:

   • Proto-oncogenes: Genes involved in promoting cell proliferation and survival. Mutations that activate proto-oncogenes can lead to uncontrolled cell growth (oncogene activation).

   • Tumor Suppressor Genes: Genes involved in inhibiting cell proliferation and promoting cell death. Mutations that inactivate tumor suppressor genes can remove inhibitory signals, promoting cancer progression.

7. Cancer Stem Cells (CSCs):

   • CSCs are a subpopulation of cancer cells with stem cell-like properties, including self-renewal and differentiation capabilities.

   • They play a critical role in tumor initiation, progression, metastasis, and therapy resistance.

8. Experimental Evidence for CSCs Driving Oncogenesis:

   • Experiments in mice have shown that only a small subset of cancer cells, enriched for CSCs, can initiate tumor formation when transplanted into immunocompromised mice.

   • These CSCs exhibit higher tumorigenic potential and are capable of recapitulating the heterogeneity of the original tumor upon transplantation.

Process of PCR (Polymerase Chain Reaction):

1. Reagents:

   • Template DNA

   • Primers (forward and reverse)

   • DNA polymerase (e.g., Taq polymerase)

   • dNTPs (deoxynucleotide triphosphates)

   • Buffer solution

2. Steps: a. Denaturation (94-98°C): Heat the reaction mixture to denature the DNA strands, separating them into single strands. b. Annealing (50-65°C): Cool the reaction to allow primers to anneal to the complementary sequences on the DNA template. c. Extension (72°C): DNA polymerase extends the primers by adding nucleotides to the 3' end, synthesizing new DNA strands complementary to the template.

3. RT-PCR (Reverse Transcription PCR):

   • Used to amplify RNA (mRNA) by first converting it to complementary DNA (cDNA) using reverse transcriptase.

   • Components include reverse transcriptase, oligo(dT) primers (to select mRNA), and PCR reagents.

   • Steps involve reverse transcription of mRNA into cDNA followed by PCR amplification of the cDNA.

4. Difference between mRNA and cDNA:

   • mRNA is the RNA molecule transcribed from DNA and contains introns and exons.

   • cDNA (complementary DNA) is synthesized from mRNA using reverse transcriptase and lacks introns, representing only exonic sequences.

5. Applications of RT-PCR:

   • Quantification of gene expression levels (transcript levels) by measuring mRNA abundance.

   • Detection of specific RNA targets such as viral RNA or mRNA transcripts.

6. Expressing Human Genes in Bacterial Cells:

   • Human genes can be expressed in bacterial cells by cloning cDNA sequences (lacking introns) into bacterial expression vectors.

   • Bacteria lack the machinery to process introns, so cDNA sequences are used to ensure proper gene expression.

7. Synthesis of Human Insulin in Bacteria:

   • RT-PCR is used to generate cDNA encoding human insulin.

   • The cDNA is cloned into a bacterial expression vector and transformed into bacterial cells.

   • Bacteria then synthesize human insulin using the cloned cDNA.

Mechanism of the Adaptive Immune System in Bacteria:

1. CRISPR-Cas System:

   • Bacteria use CRISPR-Cas system to defend against foreign genetic elements, such as bacteriophages and plasmids.

   • CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

2. CRISPR-Cas Immune Response: a. Adaptation: Bacteria incorporate short segments of foreign DNA (spacer sequences) into their own CRISPR arrays. b. Expression: CRISPR arrays are transcribed and processed into CRISPR RNA (crRNA). c. Interference: crRNA guides Cas proteins to complementary sequences in invading genetic elements. d. Destruction: Cas proteins cleave and degrade the foreign DNA, providing immunity against subsequent infections.

Repair Mechanisms for Double-Stranded DNA Breaks:

1. Non-Homologous End Joining (NHEJ):

   • NHEJ rejoins broken DNA ends directly, often leading to small insertions or deletions (indels) at the repair site.

   • It is error-prone and can result in mutations.

2. Homology-Directed Repair (HDR):

   • HDR repairs breaks using a homologous DNA template, typically the sister chromatid or homologous chromosome.

   • It ensures accurate repair by copying the intact sequence from the template, resulting in precise repair without mutations.

CRISPR-Cas9 Utilizing NHEJ and HDR:

1. NHEJ in CRISPR-Cas9:

   • CRISPR-Cas9 induces double-stranded breaks at specific genomic loci.

   • The broken ends are repaired by NHEJ, resulting in indels that disrupt the target gene's function.

2. HDR in CRISPR-Cas9:

   • HDR can be employed for precise genome editing by providing a donor DNA template along with the CRISPR-Cas9 system.

   • The template is used to repair the break, allowing for precise sequence modifications, including correction of mutations or insertion of desired sequences.

Base Editing:

• Base editing is a genome editing technique that allows for the direct conversion of one DNA base pair into another without inducing double-stranded breaks.

• It involves the fusion of a catalytically inactive Cas protein with a base-modifying enzyme, enabling precise single-base changes.

CRISPR Advancements in Immunotherapy:

• CRISPR is being used to enhance immunotherapy by:

  • Editing T cells to enhance their ability to target and kill cancer cells.

  • Knocking out genes that suppress the immune response to promote anti-tumor activity.

  • Creating personalized cancer vaccines by editing patient-derived immune cells to recognize and attack cancer-specific antigens.

Epigenetics:

1. Definition: Epigenetics refers to the study of changes in gene expression or cellular phenotype that do not involve alterations to the underlying DNA sequence. These changes are heritable and reversible and are mediated by modifications to DNA and associated proteins.

Genome vs. Epigenome:

1. Genome:

   • The genome refers to the complete set of DNA, including all genes and non-coding sequences, present in an organism's chromosomes.

   • It represents the genetic blueprint of an organism and is inherited from parents.

2. Epigenome:

   • The epigenome refers to the overall pattern of epigenetic modifications (such as DNA methylation and histone modifications) across the genome.

   • It determines which genes are active or inactive in a particular cell at a specific time and plays a crucial role in regulating gene expression.

Epigenome of Twins:

• Twins with identical genomes can have different epigenomes, especially as they age.

• Environmental factors, lifestyle choices, and random events can lead to variations in epigenetic marks between twins.

• These differences in the epigenome can result in divergent gene expression patterns and contribute to phenotypic differences between identical twins.

DNA Methylation:

• Methylation: Addition of a methyl group (CH3) to the cytosine base of DNA.

• Effects on Gene Expression: Methylation of DNA typically leads to gene silencing by preventing transcription factor binding or recruiting proteins that repress transcription.

Histone Modifications:

• Acetylation: Addition of acetyl groups to histone proteins.

• Deacetylation: Removal of acetyl groups from histone proteins.

• Effects on Gene Expression: Acetylation generally promotes gene expression by relaxing chromatin structure, while deacetylation leads to chromatin condensation and gene repression.

Heterochromatin vs. Euchromatin:

1. Heterochromatin:

   • It is densely packed and transcriptionally inactive.

   • Found in regions with highly repetitive DNA sequences and genes that are not actively transcribed.

   • Often associated with repressive histone modifications and DNA methylation.

2. Euchromatin:

   • It is less condensed and transcriptionally active.

   • Contains genes that are actively transcribed and involved in cellular functions.

   • Associated with permissive histone modifications and accessible chromatin structure.

Epigenome of Twins:

• Identical twins have the same DNA sequence but can exhibit differences in their epigenomes.

• These differences can arise due to environmental factors, lifestyle choices, and stochastic events.

• As twins age, their epigenomes can diverge further, leading to variations in gene expression and phenotypic differences.

Behavioral Genetics:

• Relationship Between Grooming, Anxiety, and Epigenetics:

  • Studies in rodents have shown that maternal grooming behavior (licking) affects the methylation of specific genes, including those related to stress response.

  • Increased maternal licking is associated with decreased anxiety-like behavior and increased expression of glucocorticoid receptors in the offspring.

  • Methylation of the promoter region of certain genes can modulate their expression, impacting behavior and stress response.

Transgenerational Epigenetic Inheritance:

• Early life experiences can induce changes in the epigenome that are passed on to offspring.

• These inherited epigenetic changes can influence the phenotype and behavior of subsequent generations.

• This phenomenon highlights the role of epigenetics in transmitting environmental effects across generations.

Effects of Maternal Care on Behavior and Epigenome:

• Studies in monkeys have shown that maternal care influences behavior and is associated with changes in the epigenome.

• Offspring that receive high levels of maternal care exhibit lower levels of anxiety and stress-related behaviors.

• Maternal care can lead to epigenetic modifications, such as changes in DNA methylation, which contribute to the observed behavioral differences.

Epigenetics in Human Diseases and Disorders:

• Epigenetic mechanisms are implicated in various human diseases, disorders, and behaviors, including cancer, neurodevelopmental disorders, and psychiatric conditions.

• Dysregulation of epigenetic processes can lead to aberrant gene expression patterns and contribute to disease pathogenesis.

Epigenetic Therapy:

• DNMT inhibitors (DNA methyltransferase inhibitors) can modulate DNA methylation patterns and are used in epigenetic therapy for certain diseases, such as cancer.

• CRISPR-dCas9 technology allows for precise manipulation of epigenetic marks, offering potential therapeutic interventions for epigenetic disorders.

Epigenetic Clock:

• The epigenetic clock refers to the correlation between chronological age and DNA methylation patterns.

• It provides a measure of biological age and can be used to assess aging-related changes and predict health outcomes.

• The epigenetic clock has implications for understanding aging processes and developing interventions to promote healthy aging.