Apoptosis

Question 1

apoptosis (3)

Answer 1

• programmed/active cell death
• requires energy, RNA and protein synthesis
• cleared by phagocytosis; no inflammation or tissue damage

Question 2

necrosis (3)

Answer 2

• passive cell death
• cells swell, membrane breaks down, contents leak, nucleus disintegrates
• inflammatory (tissue damage)

Question 3

apoptosis functions (2)

Answer 3

• multicellular development: sculpting tissues, deletion of structures, regulation of neuron number
• body’s defence against cancer

Question 4

is apoptosis evolutionarily conserved (2)

Answer 4

yes:

• occurs in all multicellular animals and plants
• stages and genes are conserved from worms and flies to mice and humans

Question 5

what phenotype is expected if engulfment is blocked

Answer 5

• accumulation of cell corpses

Question 6

characteristics of mutations affecting engulfment (3)

Answer 6

• cell death occurs at normal time, but cell corpses persist long after cells die
• DNA is less condense and not degraded in dying cells
• do not disrupt development

Question 7

how do you determine the correct position of a gene for a recessive mutation (3)

Answer 7

1. transform each gene into mutant and observe for complementation/WT phenotype
2. sequence allele of all genes in mutant; correct gene will have a mutation
3. obtain new loss of function mutants for each each gene; mutation in one of the genes should have similar phenotype to mutant phenotype

Question 8

how do you make a genomic DNA library (3)

Answer 8

1. extract DNA from cells and digest with restrictive enzyme
2. DNA fragments inserted into cloning vectors
3. transform vectors into bacterial cells; each cell will have a vector with different cDNA pieces

Question 9

cDNA library

Answer 9

• represents cDNA copies of RNA

Question 10

how to screen a DNA library (4)

Answer 10

1. blot some bacterial cells on a membrane
2. cell lysis, DNA denaturation and membrane fixing
3. add labelled probes and incubate; wash away unhybridized probes
4. identify colonies (will have hybridized DNA) that carry gene of interest

Question 11

capsases (2)

Answer 11

• cell death executors

- first synthesized as an inactive protease precursor and is later activated by proteolysis

Question 12

activation of caspases (2)

Answer 12

• self-activation: come together to form partial active sites
• by another cysteine protease/caspase

Question 13

CED-4 and APAF-1 (2)

Answer 13

• evolutionarily conserved proteins involved in regulation of caspases
• have similar domains, reflecting comparable functions in C. elegans and humans

Question 14

gain of function mutation (2)

Answer 14

• mutation that confers an enhanced activity on a protein

Question 15

characteristics of gain of function mutations (2)

Answer 15

• increased protein activity

- uncontrolled or promiscuous activity

Question 16

examples of uncontrolled or promiscuous activity (4)

Answer 16

• active without stimuli
• loss of expression control (wrong place, time, or amount)
• increase protein stability
• loss of negative regulation

Question 17

in general, loss of function mutations are… (2)

Answer 17

• recessive

- half of normal product amount is enough for normal function

Question 18

in general, gain of function mutations are… (2)

Answer 18

• dominant

- presence of the mutant version of the gene is enough to change phenotype

Question 19

how do we assess the relationship between different mutants that function in the same pathway

Answer 19

• make double mutants and observe phenotype

Question 20

interpret the statement: mutant 1 is epistatic to mutant 2

Answer 20

• mutant 1 suppresses defect in mutant 2; double mutant will have mutant 1 phenotype
• indicates that mutant 1 functions further downstream to mutant 2

Question 21

how can you determine order in mutations with the same phenotype

Answer 21

• produce or search for gf mutation in one of the genes

- then make the double mutant

Question 22

what are the 3 classes of protein functions in the conserved apoptotic family

Answer 22

• regulator
• adaptor
• effector

Question 23

pathways initiating apoptosis (3)

Answer 23

• activation of cell death from inside the cell (intrinsic/mitochondrial pathway)
• activation of cell death from outside of cell (extrinsic/death receptor pathway)
• pathways intersect at effector caspases

Question 24

intrinsic/mitochondrial pathway (5)

Answer 24

1. mitochondria release cytochrome c
2. cytochrome c activates caspase 9
3. caspase cascade
4. caspase cleavage of cellular proteins, nuclease activation
5. death

Question 25

extrinsic/death receptor pathway (4)

Answer 25

1. outside ligand binds to death receptor
2. activates pro-caspase 8
3. activates pro-execution caspase
4. death