ISOLATION OF MITOCHONDRIAL COMPONENTS

• The first successful technique for separating inner and outer membrane developed by Donald Parson.
• In this procedure mitochondria are placed in hypotonic solutions  exposed to the detergent digitonin until the outer membrane ruptures, releasing the contents of the inter membrane space into solution.
• The inner membrane, outer membranes and components of inter membrane space are then separated by centrifugation.
• Isolated inner and outer membranes can be readily distinguished from each other by electron microscopy.
• Outer membrane works like empty sacs and inner membranes from vesicles called mitoplast containing matrix material within.

• Mitoplast can be further fractionated into inner membrane and matrix components by treating with detergent Lubrol.
• Lubrol disrupts the inner membrane into small inner membrane vesicles containing F1 particles.
• Biochemical studies of isolated fractions. provides important information regarding the location of various metabolic activities of  the mitochondria.

GENOME ORGANIZATION

• The genetic evidence indicates that mitochondria contain genes distinct from those located in the nucleus.
• Mitochondrial DNA was identified on the basis of its unique density.
• The amount of mitochondrial DNA found in eukaryotic cells varies.
• In animal mitochondrial DNA-typically accounts for 0.1 to 1.0 percent of a cells total DNA content in Yeast its value is high as 15%.
• The DNA located in mitochondria exhibits several features that distinguish it from nuclear DNA.
• One major difference is that mtDNA lacks histone and hence not packaged into nucleosomes.

• Mt DNA is typically Circular.
• The mitochondrial DNA circles of animals cells are the smallest about 15,000-20,000 bp in size.
• Mt DNA of fungi and protists are larger i.e. 20,000-1,00,000bp and plant mtDNA are largest ranging from 2,00,000bp to million bp in length.
• One to the tendency of large circular DNA to break during isolation many reports suggest that plant mt DNA is linear rather than circular. Few unicellular prokaryotes such as Paramecium have linear mol of mt DNA’s.
• In animals, fungi and protists mitochondrial contains few dozens to several 100’s mol of DNA, all of which are identical.
• The first direct evidence for an association between cytoplasmic inherited tracts and mt DNA was produced by Piotr Solnimski et.al. they  demonstrated that mt DNA obtained from yeast bearing petite mutation has a different density than the mt DNA of normal yeast Petites is induced by treatment with ethidium bromide as a model system.
• These studies revealed that ethidium bromide stimulates the breakdown of mt DNA and inhibits mt DNA replication.
• Long term exposure to ethidium bromide causes the complete destruction of mt DNA, but if treatment is stopped before all the DNA has been degraded, the remaining DNA resumes replication until the total context of mt DNA reaches normal levels.
• But the new mt DNA is grossly abnormal containing few of its sequence present in the original mt DNA. The presence of abnormal mt DNA in petite yeast  strongly argues that this DNA is responsible for the abnormalities in mitochondrial structure and respiratory activity observed in petites mutants.

Isolation of Mitochondrial Components

1. Who developed the first successful technique for separating mitochondrial inner and outer membranes?
   a) Palade
   b) Donald Parson
   c) Altman
   d) Koliker
   Answer: b) Donald Parson
2. Which solution is used to rupture the outer membrane of mitochondria during isolation?
   a) Hypertonic solution
   b) Hypotonic solution
   c) Isotonic solution
   d) Alkaline solution
   Answer: b) Hypotonic solution
3. What detergent is used in the mitochondrial isolation process to rupture the outer membrane?
   a) Digitonin
   b) SDS
   c) Triton X-100
   d) Tween-20
   Answer: a) Digitonin
4. Which technique is used to separate mitochondrial inner and outer membranes?
   a) Spectrophotometry
   b) Centrifugation
   c) Gel electrophoresis
   d) Chromatography
   Answer: b) Centrifugation
5. What is the term for vesicles containing mitochondrial matrix material?
   a) Cristae
   b) Mitoplast
   c) Lysosome
   d) Ribosomes
   Answer: b) Mitoplast
6. Which detergent is used to disrupt the inner membrane of mitochondria?
   a) SDS
   b) Lubrol
   c) Digitonin
   d) Triton X-100
   Answer: b) Lubrol
7. What is released into the solution after the rupture of the outer mitochondrial membrane?
   a) Matrix contents
   b) Intermembrane space components
   c) Cristae
   d) F1 particles
   Answer: b) Intermembrane space components
8. Which part of mitochondria resembles empty sacs under electron microscopy?
   a) Inner membrane
   b) Outer membrane
   c) Cristae
   d) Matrix
   Answer: b) Outer membrane
9. F1 particles are associated with which mitochondrial membrane?
   a) Outer membrane
   b) Inner membrane
   c) Intermembrane space
   d) Matrix
   Answer: b) Inner membrane
10. What percentage of mitochondrial inner membrane proteins are oxysomes?
    a) 5%
    b) 10%
    c) 15%
    d) 20%
    Answer: c) 15%

Genome Organization

11. Mitochondrial DNA was identified based on its unique _______.
    a) Shape
    b) Density
    c) Color
    d) Size
    Answer: b) Density
12. Which type of DNA is found in mitochondria?
    a) Linear
    b) Circular
    c) Branched
    d) None of the above
    Answer: b) Circular
13. Which of the following is absent in mitochondrial DNA?
    a) Exons
    b) Histones
    c) tRNA genes
    d) Ribosomal RNA
    Answer: b) Histones
14. The percentage of mitochondrial DNA in a eukaryotic cell’s total DNA content is around:
    a) 0.01 – 0.1%
    b) 0.1 – 1.0%
    c) 1 – 5%
    d) 10 – 15%
    Answer: b) 0.1 – 1.0%
15. In yeast, mitochondrial DNA content can be as high as:
    a) 1%
    b) 5%
    c) 15%
    d) 25%
    Answer: c) 15%
16. The smallest known mitochondrial genome is found in:
    a) Humans
    b) Yeast
    c) Plasmodium falciparum
    d) Paramecium
    Answer: c) Plasmodium falciparum
17. The size of the human mitochondrial genome is:
    a) 6 kb
    b) 16.5 kb
    c) 78 kb
    d) 200 kb
    Answer: b) 16.5 kb
18. In plant cells, the size of mitochondrial DNA is:
    a) 2 kb
    b) 16 kb
    c) 200 kb – 1 million bp
    d) 500 bp
    Answer: c) 200 kb – 1 million bp
19. The mtDNA genome in most eukaryotic cells is inherited from:
    a) Both parents
    b) Only the father
    c) Only the mother
    d) Randomly from either parent
    Answer: c) Only the mother
20. What enzyme is required for mitochondrial DNA replication?
    a) DNA polymerase α
    b) DNA polymerase β
    c) DNA polymerase γ
    d) DNA polymerase δ
    Answer: c) DNA polymerase γ

Mitochondrial Mutations & Studies

21. Petite mutants were first studied in:
    a) Humans
    b) Bacteria
    c) Yeast
    d) Plants
    Answer: c) Yeast
22. Who provided direct evidence for cytoplasmic inheritance in yeast?
    a) Palade
    b) Donald Parson
    c) Piotr Solnimski
    d) Fleming
    Answer: c) Piotr Solnimski
23. Ethidium bromide exposure in yeast leads to:
    a) Increased ATP production
    b) mtDNA degradation
    c) More mitochondria
    d) Increased oxidative stress
    Answer: b) mtDNA degradation
24. The difference in density of mitochondrial DNA in petite yeast mutants is due to:
    a) Increased mutations
    b) Reduced nucleotide content
    c) Different protein-coding sequences
    d) All of the above
    Answer: d) All of the above
25. What is the function of cytochrome c in mitochondria?
    a) ATP synthesis
    b) DNA replication
    c) Electron transport chain
    d) Protein synthesis
    Answer: c) Electron transport chain
26. The majority of mitochondrial proteins are coded by:
    a) mtDNA
    b) Nuclear DNA
    c) Both mtDNA and nuclear DNA
    d) Ribosomes
    Answer: b) Nuclear DNA
27. The process of transferring electrons within mitochondria is called:
    a) DNA replication
    b) Glycolysis
    c) Electron transport chain
    d) Fermentation
    Answer: c) Electron transport chain
28. Which enzyme is NOT involved in oxidative phosphorylation?
    a) ATP synthase
    b) Cytochrome c oxidase
    c) Hexokinase
    d) NADH dehydrogenase
    Answer: c) Hexokinase
29. Which cellular process is most affected by mitochondrial mutations?
    a) DNA synthesis
    b) ATP production
    c) Lipid synthesis
    d) Cell signaling
    Answer: b) ATP production
30. A major function of mitochondria is:
    a) Protein degradation
    b) Energy production
    c) Cell membrane synthesis
    d) Detoxification
    Answer: b) Energy production

Mitochondrial Structure & Function

31. The mitochondria are also known as:
    a) Powerhouse of the cell
    b) Control center of the cell
    c) Protein factory of the cell
    d) Digestive system of the cell
    Answer: a) Powerhouse of the cell
32. The space between the inner and outer mitochondrial membrane is called:
    a) Mitochondrial matrix
    b) Peri-mitochondrial space
    c) Cristae
    d) Nucleoid
    Answer: b) Peri-mitochondrial space
33. Which enzyme is responsible for ATP synthesis in mitochondria?
    a) Hexokinase
    b) ATPase
    c) Succinyl-CoA synthetase
    d) Pyruvate kinase
    Answer: b) ATPase
34. The infoldings of the inner mitochondrial membrane are called:
    a) Thylakoids
    b) Cristae
    c) Grana
    d) Matrix
    Answer: b) Cristae
35. Mitochondria play a crucial role in:
    a) Glycolysis
    b) DNA transcription
    c) Oxidative phosphorylation
    d) Lipid synthesis
    Answer: c) Oxidative phosphorylation
36. The inner mitochondrial membrane is impermeable to:
    a) Oxygen
    b) CO₂
    c) Protons
    d) Water
    Answer: c) Protons
37. Which of the following occurs inside the mitochondrial matrix?
    a) Glycolysis
    b) Electron Transport Chain
    c) Citric Acid Cycle (Krebs Cycle)
    d) Photosynthesis
    Answer: c) Citric Acid Cycle (Krebs Cycle)
38. Which molecule is the final electron acceptor in the Electron Transport Chain?
    a) NAD+
    b) FAD
    c) Oxygen
    d) Water
    Answer: c) Oxygen
39. The enzyme succinate dehydrogenase is found in which part of mitochondria?
    a) Inner membrane
    b) Outer membrane
    c) Matrix
    d) Intermembrane space
    Answer: a) Inner membrane
40. Mitochondria participate in cell apoptosis by releasing which protein?
    a) Cytochrome b
    b) Cytochrome c
    c) ATP synthase
    d) Ubiquinone
    Answer: b) Cytochrome c

Mitochondrial Genetics & Mutations

41. What is the size of the mitochondrial genome in humans?
    a) 6 kb
    b) 16.5 kb
    c) 100 kb
    d) 1 million bp
    Answer: b) 16.5 kb
42. Mitochondrial DNA replication occurs during which cell cycle phase?
    a) G1 phase
    b) G2 phase
    c) S phase
    d) M phase
    Answer: b) G2 phase
43. The non-Mendelian inheritance of mitochondria is called:
    a) Dominant inheritance
    b) Recessive inheritance
    c) Cytoplasmic inheritance
    d) Epigenetic inheritance
    Answer: c) Cytoplasmic inheritance
44. The presence of multiple types of mitochondrial DNA in a single cell is called:
    a) Homoplasmy
    b) Heteroplasmy
    c) Polyploidy
    d) Aneuploidy
    Answer: b) Heteroplasmy
45. The mitochondrial genome is rich in which type of base pair?
    a) A-T
    b) G-C
    c) T-G
    d) C-T
    Answer: b) G-C
46. The smallest known mitochondrial genome belongs to:
    a) Yeast
    b) Humans
    c) Plasmodium falciparum
    d) E. coli
    Answer: c) Plasmodium falciparum
47. Which of the following inhibits mitochondrial protein synthesis?
    a) Penicillin
    b) Chloramphenicol
    c) Streptomycin
    d) Rifampicin
    Answer: b) Chloramphenicol
48. What is the major function of mitochondrial tRNA?
    a) DNA replication
    b) Protein synthesis
    c) ATP synthesis
    d) Lipid metabolism
    Answer: b) Protein synthesis
49. The majority of mitochondrial proteins are synthesized by:
    a) Mitochondrial ribosomes
    b) Cytoplasmic ribosomes
    c) Golgi apparatus
    d) Peroxisomes
    Answer: b) Cytoplasmic ribosomes
50. Mitochondrial mutations are commonly associated with:
    a) Cancer
    b) Neurodegenerative diseases
    c) Metabolic disorders
    d) All of the above
    Answer: d) All of the above