Organism Dev Flashcards
What is forward genetics vs backward genetics?
Forward genetics → Starting from the phenotype (weird) and looking for what changes occurred in the genotype
Reversed genetics → Starting from changes in the genotype and seeing how it affects the phenotype
What are 2 important characteristics of model organisms?
- Mimic specific aspects of human biology
- Are (comparatively to humans) easy to work with
What are the main 5 eukaryotic model organisms?
- S.cerevisiae (yeast)
- C. elegans
- Drosophila melanogaster
- Danio rerio (zebrafish)
- Mus musculus (house mouse)
From 1 → 5: Get more like humans, but get more difficult to grow/work with
*Diverged from human more and more recently in history/development
What are the characteristics of S. cerevisiae?
(generation time, advantages, disadvantages)
- Eukaryotic, UNICELLULAR fungus
- Generation time: 2-3 hours
- Can exist as haploid or diploid
- Can reproduce sexually and asexually
- Can be frozen and revived (easy)
What is measured when we talk about the generation time?
It is the time between when the embryo is conceived to when the offspring is capable of reproducing itself (mature enough), of conceiving an embryo
What does the life cycle/reproduction cycle of S. cerevisiae look like?
- Haploid go through asexual reproduction (budding) → haploid offspring
- Haploid can mate to make diploid offspring → mitosis → back to haploid (x2)
- Diploid can bud and produce diploid offspring
What are the advatanges of yeasts in their haploid form vs in their diploid form?
Haploid form → easy to study gene effect (direct)
Diploid form → study gene interaction, patterns of dominance, trans-acting, etc.
What are the characteristics of C.elegans?
(generation time, advantages, disadvantages)
- Invertebrate animal, multicellular
- Generation time: 3 days → 300 progeny (eggs)
- Extremly simple → translucent
- Can trace fate of each cell (1090 total)
- Can be frozen and revived
2 sexes: male (XO) & hermaphrodite (XX):
- Hermaphrodite can self-fertilize → make mostly hermaphrodites
- Can also mate with males → different combinations of genes
What does the life cycle of C. elegans look like?
Normal cycle:
L1 → 12h + 8h + 8h → L4 → 18h → Reproductive adult (can lay eggs) → embryo → 14h → L1
Dauer stage → if exposed to a tough envrionment → hibernation → can later develop and lay eggs when get out of Dauer stage
take ~ 13h from L1 → Dauer (can then stay several months) → L4 (out of Dauer)
What are the characteristics of D. melanogaster?
(generation time, advantages, disadvantages)
- Invertebrate animal, mutlicellular
- Generation time: 10 days → 100 progeny
- More complex than C. elegans
- Share 75% of human disease-causing genes (how they affect specific cell-types)
- Very well studied, many genetic tools
What are the characteristics of Danio rerio?
(generation time, advantages, disadvantages)
Danio rerio = zebrafish
- Vetebrate animal, multicellular → CNS closer to human
- Generation time: 2-3 months → 200 eggs
- Optically translucent embryo & larva
- Relatively simple & inexpensive to maintain (need aquarium)
- Easily treated with small molecules for drug & toxicity screens
What are the characteristics of Mus musculus?
(generation time, advantages, disadvantages)
- Vertebrate MAMMAL
- Generation time: 3 months → 2-12 pups
- Small, easy to house (for a mammal)
- Commonly used to study human biology, perform preclinical testing
**Mice are not always perfect models for human, there are some major differences!!
What is special about Axolotl mexicanum as an emerging model organism?
In can regenerate its limbs
What is special about Planaria as a model organism?
It can regenerate its whole body from any cut part (full of stem cells)
What are the 3 main steps of a forward genetic screen?
- Perturb a lot of genes (randomly or systematically)
ex: chemical mutagen - Look for a specific phenotype
ex: The organism dies, changes in some specific way - Figure out which gene you mutated
How was cell cycle of S.cerevisiae explored using a forward genetic screen?
Why is S. cerevisiae used to study cell cycle?
We can visualize different stages of the cell cycle easily:
G1 phase → tiny bud
S phase → DNA being replicated and pushed into the bud
G2/M phase → bud separation → 2 cells
What is the mechanisms and the importance of temperature sensitive mutants?
Problem: The cell division cycle mutants of interest couldn’t be cultured and replicated because they couldn’t go through the cell cycle
Solution: Temperature sensitive mutants → At low permissive temperatures, the yeast can replicate/divide
At high restrictive temperatures, the mutant phenotype can be observed (blocked at a specific stage of the cell cycle) → can go back to dividing when brought back to lower temperatures
*So mutants can be expanded and studied
What is replica plating used for?
- Put different colonies in aggregates in a master plate
- Stamp the colonies on a sterile velveteen (sticky)
- Stamp the colonies on different plates at different temperatures
- Can see which mutants grow or not at different temperature and compare since all the plates have the same organization
Used to isolate temperature sensitive mutants
What process is affected in cdc1 mutant yeasts?
Cells have a small bud, but won’t start replication, bud stays very small (stops in G1)
Functional gene product: Putative metallophosphodieasterase → involved making cell wall (not helpful for humans)
What process is affected in cdc2 mutant yeasts?
Mutants can form a bud, can see the start of DNA replication, but doesn’t complete it, stops in S phase
Functional gene product: DNA polymerase delta catalytic subunit
What process is affected in cdc3 mutant yeasts?
Cdc3 mutants stop in late stages of mitosis, division of the buds can happen to a certain extent, but the cells can’t separate 100%
Functional gene product: Septin family member, involved in cytokinesis
How did researchers confirm that 2 yeasts with the same phenotype had a mutation in the same gene?
By complementation test:
If mating the 2 haploid yeasts together → diploid mutant → same gene
If matin the 2 haploid yeasts together → diploid WT → different genes bc the WT copy of each gene complemented
*2nd stage of the forward screen
How did researchers proceed to identify which was the mutant gene in the yeast after having identified the different cell cycle mutants? (cdc)
*Final stage of a forward genetic screen
Try to rescue the mutant with different DNA plasmids:
- Yeast dsDNA → cleaved w/restriction endonuclease
- Thousands of genomic DNA fragments
- DNA fragment inserted into plasmids
- Introduce into multiple bacterias → Yeast genomic DNA library
- Introduce different plasmids into yeast → test which plasmids rescue the mutant
Sequence plasmid that rescues → gene that is mutated in the mutant yeast
*plasmids are WT
How did researchers assess if some human genes were related to the mutated cdc genes in yeast?
Made cDNA plasmids from human mRNA → test which plasmid rescuse the yeast cdc mutant
What was the human version of cdc28 identified through forward genetic screening?
cdc2 (S. pombe) = cdc28 (S. cerevisiae) = Cdk1 (human)
*Very conserved in cell cycle regulation
Which model organism was used to analyze apoptotic pathways?
Why?
C. elegans
- Every embryo develops with the same pattern of cell division & migration
- Adults worms have exactly 959 cells generated in the exact same way
1090 cells formed during embryogenesis → 131 die, the exact same always
*Only disadvantage, when cells die, they are difficult to see as they are engulfed very quickly
What was seen in the ced-1 C. elegan mutant?
In ced-1, they could see spots of dead cells → cells could dye, but could not be engulfed
Can use that to see if cell death is affected by other mutants because you can visualize cell death → They did a suppressor screen to find mutants in the ced-1 background
What was seen in the ced-1 and ced-3 double mutants in C. elegans?
They could no longer see visible corps → no cell death → means cell death is an active process as it is regulated/induced by specific genes
What is specific of Egl-1 C. elegans mutants? What explains it?
Egl-1 can make eggs but can’t lay them → fat worms
It is explained by the lack of Hermaphrodite-specific neurons (HSN) which are required for cell death → a bit upreagulated cell death and HSN are more sensitive
*Used to do supressor screens