Part 6: Apoptosis Flashcards
(29 cards)
What is apoptosis
Programmed cell death, active and requires energy
Removed by autophagy and does not cause tissue inflammation
Conserved and occurs in the same order in all species
What is necrosis
Passive cell death, inflammatory
Function of apoptosis
- Multicellular development (tissue sculpture, structure deletion and neuron number regulation)
- Defense against cancer
Stages of apoptosis
Cell death initiation
Engulfment by scavenger cell
Removal
In the loss of function nuc-1 mutant, cell death occurs at normal time, but DNA in the death cells fail to be degraded.What is the normal function of Nuc-1?
a) To promote DNA degradation
b) To prevent DNA degradation
Promote DNA degradation
What phenotype do you expect if engulfment is blocked?
Expect to see cell corpse in the tissue
Describe phenotype of ced-1 and ced-2 mutants
Cell corpse persist in cell, DNA is less condensed and not degraded, no disruption of development
What is supressor screen used for
To identify mutations that reverse the effect of another mutation, that is genes that are upstream
Supressor of ced-1
Ced-3 and ced-4, no cell death when heterozygous with ced-1
You isolated two cell death mutants, which abolish both the embryonic and post embryonic cell death. Both mutants do not disrupt other developmental process. What are the normal functions of the corresponding genes?
a) To promote cell death
b) To prevent cell death
c) Not yet enough information to decide
To promote cell death
Describe the relationship between cell death receptors
Ced-4 regulates ced-3, both are upstream of ced-1 and ced-2, which are upstream of nuc-1
The recessive ced-3 mutation was mapped to a region between markers unc-30 and nP33. This region is about 60 kb, which contains 15 genes.
What is positional cloning
Make clones of different sizes complementary to the DNA sequence and identify those that are complementary to the gene which position is to be found
Use the genomic clone S3 to screen a cDNA library, a 2.5 kb cDNA clone of Ced-3 was isolated. Sequencing the clone revealed that it encoded a protein with 503 amino acids. What do you think the researchers immediately did to find out the possible functions of the protein?
a) Use RT-PCR to determine the expression level of Ced-3.
b) Use in situ hybridization to determine the expression pattern of Ced-3.
c) Create transgenic animals carrying a promoter:GUS construct and use it to determine the expression pattern of Ced-3.
d) Carry out a Blast search to find proteins similar of Ced-3.
e) Purify Ced-3 protein and analyze its biochemical function.
Blast search, want to see if similar genes were already studied
What is a caspase
Cysteine protease (cysteine-dependent aspartate-directed proteases) , synthesized as precursor and activated by proteolysis at Aspartate residues
Two mechanisms of caspase activation
1) Self-activation: two caspase activate when come into contact
2) Activation by another caspase: residual amount of caspase is able to activate other precursor enzyme
How does insertional tagging work?
Insert a Tc4 tag into gene, DNA is digested and hybridized to a probe, positive Tc4 probe must have a gene of interest
What is a ced-9 mutation
Dominant mutation responsible for death cell, no cell corpse found in ced-1/ced-9 mutants
You isolated a dominant (gain of function) cell death mutant, which suppresses both the embryonic and post embryonic cell death. The mutant does not disrupt other developmental process. What is the normal function of the corresponding gene?
a) To promote cell death
b) To prevent cell death.
c) Not yet enough information to decide
To promote cell death
What is a GOF mutation?
Mutation with increased or uncontrolled and promiscous protein activity:
- No stimuli required
- No expression control (wrong place, time, increased amount)
- Increased stability
- No negative regulation
What is a general difference between GOF and LOF?
LOF is usually recessive, while GOF is dominant (one copy is enough)
When is LOF not recessive?
Haploinsufficient with intermediate phenotype or dominant negative, interferes with a normal allele
You isolated a dominant (gain of function) cell death mutant, which suppresses both the embryonic and post embryonic cell death. The mutant does not disrupt other developmental process. What is the most likely function of the wild type gene?
a) To promote cell death
b) To prevent cell death
To prevent cell death
What’s your prediction on the mutant phenotype of the loss of function allele of ced-9?
Promote cell death
