Bio 316 Exam 2

In forward genetics, what is the first step?

Start with a phenotype of interest

What is the purpose of treating organisms with a mutagen in forward genetics?

To cause random DNA changes and search for mutants

Which of the following is an example of a forward genetics approach?

A) Disrupting ADE2 with CRISPR

B) Searching for uncoordinated worms to study axon guidance

C) Deleting a gene and observing no change in phenotype

D) Designing a specific sequence mutation

Searching for uncoordinated worms to study axon guidance

In reverse genetics, researchers begin by...

Selecting a gene of interest

Which method is commonly used in reverse genetics to disrupt gene function?

A) Mutagenesis by UV light

B) Random mutation selection

C) Gene deletion or insertion

D) Observation of spontaneous mutants

Gene deletion or insertion

Disrupting the ADE2 gene by CRISPR in yeast is an example of:

Reverse Genetics

What is the final goal in a forward genetics experiment?

To identify the gene responsible for a phenotype

Petite mutants in yeast were identified by using which genetic approach?

Forward genetics

List steps of forward genetics

- phenotype of interest

- treat with mutagen

- select mutants

- identify gene

List steps of reverse genetics

- gene of interest

- disrupt gene (change sequence through insertion or deletion)

- observe the phenotype

What type of phenotype do "petite" yeast mutants display?

slow growth = tiny colonies

What is the mutagen used to treat S. cerevisiae in our experiment?

isopropanol

What causes the "petite" phenotype in yeast?

loss of mitochondrial function (mtDNA)

Which of the following best describes the goal after identifying petite mutants?

A) Introduce new plasmids

B) Characterize and draw conclusions about respiration

C) Return them to wild-type phenotype

D) Overexpress nuclear genes

Characterize and draw conclusions about respiration

Why might a "petite" mutant be classified as rho⁰?

It completely lacks mitochondrial DNA

Which process best describes our experiment's overall method?

Forward genetics starting from a phenotype

What question does our experiment aim to answer about the petite mutants?

Are they respiratory deficient?

List the steps of yeast mutagenesis

- phenotype of interest

- treat with isopropanol

- identify petite mutants

- characterize mutants

- draw conclusions

What does it mean when we say mutants are respiratory deficient?

they cannot convert glucose into energy (ATP)

What type of nucleic acid synthesis does ethidium bromide selectively inhibit?

mtDNA and mtRNA

What is a major cellular effect of ethidium bromide?

A) Enhances ribosome production

B) Induces mtDNA fragmentation

C) Repairs broken DNA

D) Activates glycolysis

Induces mtDNA fragmentation

What enzyme activity leads to the fragmentation of mtDNA after ethidium bromide exposure?

Endogenous nucleases

Which of the following is not affected by ethidium bromide at typical mutagenic concentrations?

A) Mitochondrial RNA synthesis

B) Mitochondrial DNA synthesis

C) Nuclear DNA synthesis

D) Mitochondrial genome structure

Nuclear DNA synthesis

What is the general shape of mitochondria?

Football shaped

How many mitochondria can be found in a typical cell?

A) 1-5

B) 10-50

C) 100-200

D) 1-1000s

1-1000s

What are the two membranes of the mitochondria?

inner and outer membranes (phospholipid bilayers)

What is located in the inner mitochondrial membrane that is crucial for ATP production?

Protein complexes for ATP synthesis (oxidative phosphorylation)

What occurs in the intermembrane space of mitochondria?

Proton (H+) pumping

Which of the following is not found in the mitochondrial matrix?

A) mtDNA

B) Ribosomes

C) RNA

D) Golgi vesicles

Golgi vesicles

Why are mitochondria referred to as the "powerhouse of the cell"?

A) They regulate the cell cycle

B) They synthesize glucose

C) They produce most of the cell's ATP through respiration

D) They digest macromolecules

They produce most of the cell's ATP through respiration

Where does glycolysis occur during cellular respiration?

Cytosol

What is the net ATP gain from the aerobic stage of respiration?

34 ATP

What is the anaerobic stage of respiration? Aerobic stage?

- glycolysis

- oxidative phosphorylation

What is the basic pathway of glycolysis (reactant --> product)?

Glucose --> Glucose-6-P --> 2 Pyruvate

What is the basic pathway of oxidative phosphorylation (reactant --> product)?

2 pyruvate --> CO2 + H2O + 34 ATP

What is the role of oxygen in the electron transport chain (ETC)?

electron acceptor

What happens to protons in the ETC as electrons are passed along protein complexes?

Protons are pumped into the intermembrane space

Name the three ETC complexes in order from left to right

- NADH dehydrogenase complex

- Cytochrome (b-c1) c reductase complex

- Cytochrome c oxidase complex

What allows protons to re-enter the mitochondrial matrix, driving ATP synthesis?

ATP synthase

What causes the conformational change in ETC proteins that allows protons to be pumped?

electron flow

Proton pumping does what to pH and charge?

- increases pH

- leaves negative charge

What is the main consequence of chemical mutagenesis affecting genes for carrier proteins in yeast mitochondria?

A) Increased ATP production

B) Complete shutdown of glycolysis

C) Only 2 ATP produced per cycle

D) More rapid cell division

Only 2 ATP produced per cycle

Compared to wild-type yeast, what happens to the growth of petite mutants?

They grow slower

What type of energy production is lost when mitochondrial carrier protein genes are disrupted?

Oxidative Phosphorylation

What defines petite mutants of yeast?A) Overproduction of ATP

B) Enhanced mitochondrial DNA

C) Respiratory deficiency due to loss of mitochondrial function

D) High resistance to mutagens

Respiratory deficiency due to loss of mitochondrial function

What does the term rho⁰ refer to in yeast?

A) Mutants with excess mitochondrial DNA

B) Mutants with no nuclear DNA

C) Mutants with complete loss of mitochondrial DNA

D) Mutants that only use fermentation

Mutants with complete loss of mitochondrial DNA

How do colonies formed by petite mutants compare to those of wild-type yeast?

smaller

If the electron transport chain is not functional, how can cells still generate ATP?

through fermentation in glycolysis

What is the difference between petite mutants and wild-type regarding ATP production?

- petite mutants only produce 2 ATP per cycle

- WT produce 34 ATP per cycle

What is the difference between petite mutants and wild-type regarding growth?

- petite mutants grow slower

- WT grow faster

What is the difference between petite mutants and wild-type regarding oxidative phosphorylation?

- Petite mutants do not have this

- WT does have this

What is one unique feature of mitochondria related to DNA?

A) They share DNA with chloroplasts

B) They contain their own genome

C) They lack any form of DNA

D) Their DNA is stored in the nucleus

They contain their own genome

How is mitochondrial DNA (mtDNA) different from nuclear DNA?

A) It is identical in sequence

B) It is a temporary copy of nuclear DNA

C) It is distinct and separate from nuclear DNA

D) It only encodes ribosomal RNA

It is distinct and separate from nuclear DNA

What does mitochondrial DNA encode?

A) All proteins needed for mitochondrial function

B) Some proteins used in mitochondrial processes

C) Only glycolysis-related proteins

D) Proteins for nuclear division

Some proteins used in mitochondrial processes

Where are most mitochondrial proteins encoded?

A) In the mitochondrial genome

B) On ribosomes inside the mitochondrion

C) By nuclear genes and imported from the cytoplasm

D) In the lysosome

By nuclear genes and imported from the cytoplasm

Which mitochondrial complex has the most subunits encoded by the mitochondrial genome?

A) Cytochrome b-c1 complex

B) NADH dehydrogenase

C) ATP synthase complex

D) Cytochrome c oxidase

NADH dehydrogenase (7 subunits)

How many subunits of the cytochrome b-c1 complex are encoded by the mitochondrial genome?

1

How many subunits of the cytochrome c oxidase complex come from the mitochondrial genome?

3

The ATP synthase complex includes how many subunits encoded by mtDNA?

2

How might DNA staining patterns help identify petite mutants?

A) They show enhanced nuclear DNA expression

B) They show decreased or absent mitochondrial DNA staining

C) They glow green under UV light

D) They only stain if glucose is present

They show decreased or absent mitochondrial DNA staining

Why do petite mutants form white colonies instead of red?

They lack oxidative phosphorylation

What color are wild-type yeast colonies under normal conditions with functional mitochondria?

red

How does isopropanol primarily affect mitochondria?

A) By mutating mtDNA directly

B) By interfering with glycolysis

C) By disrupting the mitochondrial membrane

D) By increasing ATP production

By disrupting the mitochondrial membrane

What is an indirect effect of mitochondrial membrane disruption caused by isopropanol?

A) Increase in mitochondrial protein synthesis

B) Enhanced nuclear DNA replication

C) Reduction of mitochondrial DNA

D) Direct mutation of mtRNA

Reduction of mitochondrial DNA

Which of the following statements is TRUE regarding isopropanol's impact on mitochondria?

A) It deletes nuclear genes

B) It increases mtDNA expression

C) It does not directly affect mitochondrial DNA

D) It permanently fixes mitochondrial membrane integrity

It does not directly affect mitochondrial DNA

Why is mtDNA important in yeast cells?

A) It helps in DNA replication

B) It encodes subunits of the electron transport chain

C) It forms the cell wall

D) It produces glucose

It encodes subunits of the electron transport chain

What is the energy currency of the cell?

ATP

What are the two ways of energy production from sugar?

- direct burning

- stepwise oxidation

Where is energy stored in the ATP molecule?

bond between the second and third phosphates

What happens when ATP is converted into ADP?

Energy is released

How does ADP become ATP?

by gaining a third phosphate group (gamma)

What happens when ADP is converted to ADP?

energy is stored

Where does oxidative phosphorylation occur in yeast cells?

mitochondria

What happens to electrons after they pass through the electron transport chain in yeast?

They combine with oxygen to form water

What is the ATP investment phase of glycolysis?

2 molecules ATP are consumed

What is the gross production of ATP and NADH in glycolysis?

4 ATP, 2 NADH + H+

What is the net production of ATP and NADH in glycolysis?

2 ATP, 2 NADH + H+

What is the final product of glycolysis for each molecule of glucose?

2 pyruvate

How does yeast compensate for the lack of mitochondria under anaerobic conditions?

A) It produces 2 pyruvates and converts them directly into ATP

B) It undergoes anaerobic fermentation, producing 2 CO2 and 2 ethanol

C) It uses light to produce ATP

D) It converts glucose into oxygen and carbon dioxide

It undergoes anaerobic fermentation, producing 2 CO2 and 2 ethanol

Why is anaerobic fermentation important for yeast?

A) It allows for the regeneration of NAD+, enabling glycolysis to continue

B) It produces more ATP than aerobic respiration

C) It synthesizes new mitochondria

D) It converts glucose into oxygen

It allows for the regeneration of NAD+, enabling glycolysis to continue

What is the product of anaerobic fermentation in muscle cells?

2 lactates

How does anaerobic fermentation in muscle cells differ from yeast fermentation?

A) Yeast produces lactates while muscle cells produce ethanol

B) Muscle cells convert 2 pyruvates into lactates, while yeast converts them into ethanol and CO2

C) Muscle cells produce more ATP than yeast cells

D) Both produce the same products

Muscle cells convert 2 pyruvates into lactates, while yeast converts them into ethanol and CO2

What are the reactants and products of fermentation for yeast?

2 pyruvate -> 2CO2 + 2 ethanol

What happens to pyruvate in eukaryotic mitochondria?

It is metabolized into acetyl CoA

In which complex does the conversion of pyruvate to acetyl CoA occur?

Pyruvate dehydrogenase complex

What does the pyruvate dehydrogenase complex do to pyruvate?

strips pyruvate of hydrogens

What is produced from one turn of the citric acid cycle?

3 NADH, 1 GTP, 1 FADH2, 2 CO2

Why is the citric acid cycle important for oxidative phosphorylation?

A) It generates ATP directly

B) It produces NADH and FADH2, which donate electrons to the electron transport chain

C) It synthesizes glucose for energy

D) It produces oxygen for cellular respiration

It produces NADH and FADH2, which donate electrons to the electron transport chain

How does NADH contribute to cellular respiration?

donates electrons to the electron transport chain

What is the final electron acceptor in the electron transport chain?

oxygen (O2)

What two energy molecules donate electrons to the ETC?

NADH and FADH2

Why do mitochondria have their own ribosomes?

they have their own DNA which needs to be encoded into protein

What two molecules can be converted into acetyl CoA in the mitochondria?

pyruvate and fatty acids

What is the role of ATP synthase in energy production?

A) It pumps protons into the intermembrane space

B) It oxidizes glucose into CO2

C) It allows H⁺ to pass through and synthesizes ATP from ADP

D) It transports electrons from complex I to complex II

It allows H⁺ to pass through and synthesizes ATP from ADP

What is the fate of water in the electron transport chain?

A) It is used as a reactant

B) It is stored for later energy use

C) It is a byproduct of the process

D) It initiates ATP synthesis

It is a byproduct of the process

What molecule transfers electrons from Complex I to Complex II?

Ubiquinone

Which molecule transfers electrons from Complex II to Complex III?

Cytochrome c

Why is Complex III called the Cytochrome Oxidase Complex?

oxidizes cytochrome c during electron transfer

How efficient is fermentation (%)?

How efficient is aerobic respiration?

- 2.1%

- 33%

What is the role of glycerol in testing petite mutants?

A) It activates fermentation

B) It allows growth without mitochondria

C) It tests mitochondrial function by requiring oxidative phosphorylation

D) It stains colonies red

It tests mitochondrial function by requiring oxidative phosphorylation