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RICHA MCDB 138 (MT 1)
Terms in this set (62)
How do you know all cells contain the same genetic information?
Two experiments: blastomere isolation and nuclear transplantation
Blastomere isolation experiment
Shows blastomeres are totipotent
1. Separate the sea urchin embryo at two-cell state into two blastomeres.
2. Allow each to develop - find that both develop into whole organisms (that are half-sized).
3. Conclusion: blastomeres must have totipotent cells
Nuclear transplantation experiment
Shows that any fully differentiated cell has all the genetic material of an organism
1. Take the nucleus out of a differentiated cells (like a fibroblast).
2. Enucleate an oocyte (using UV irradiation or a needle).
3. Insert the differentiated nucleus into the enucleated oocyte.
4. Find that it develops into a whole organism.
What differentiated cells don't have all genetic information?
- haploid gametes
- blood cells
Totipotency, Pluripotency, Unipotency
1. totipotency: can develop into a whole organism
2. pluripotency: can develop into one of several different cell/tissue types
3. unipotent: can develop into one cell type
At what point does a blastomere lose totipotency?
after the 2-8 cell stage
How was Dolly created?
somatic cell nuclear transplant
What did Dolly prove to the world?
that mammalian differentiated cells are totipotent
How do you make iPS cells?
plate differentiated cells with several transcription factors that drive genome-wide demethylation (introduce TFs with viruses)
What are some ways you can see differential gene expression in embryos?
in situ hybridization, antibody staining
What are the categories of cell-cell interactions?
- short range (cell surface factors)
- long range (secreted factors)
What is a fate map?
stain different layers of tissues in the beginning stages of embryonic development and observe what cell type that tissue layer develops into
How can you tell if a cell/group of cells has been determined?
1. Transplant a group of cells from one tissue layer into another tissue layer.
2. If the transplanted cells turn into the same type as their new surrounding cells, they were not determined.
3. If the transplanted cells turn into the same type as their old surrounding cells, they were determined.
What are the two steps to studying how genes affect development?
1. Find which genes affect development (via genetic screens)
2. Find how each gene affects development.
What are some methods to study how genes act in development?
1. observational: where and when is the gene expressed
2. biochemically shut off or induce a product of gene expression
3. transplant the gene to a different part of the body where it is not normally expressed
What are some invertebrate model organisms and their advantages?
- nematode: short life cycle, clear bodies
- sea urchin
What are some vertebrate model organisms and their advantages?
When and where do germ cells develop?
during early embryonic development, long before gonads are made; migrate to gonads later. Mammal eggs are not derived from a stem cell population.
What processes take place in spermatogenesis?
compaction of nuclear material (no histones), loose the ribosomes and mRNA, build microtubule-based flagellum.
Name the structures in the sperm.
- acrosomal vesicle (from Golgi)
- microtubule axoneme with dynein arms and ATPases
- nuclear material
What materials does the egg provide?
- egg shell/envelope
- cytoplasm (contains DNA, mitochondria, determinants, mRNA, ribosomes, centrosome, basically everything the sperm doesn't have)
What processes take place in oogenesis?
- synthesize proteins
- increase mitochondria and cell membrane
- make ribosomes and mRNA
- in some species, take up yolk and localize cytoplasmic determinants
What are nurse cells, where are they found, and why are they helpful?
Nurse cells are germline stem cells that don't develop into oocytes. They get smaller as the oocyte gets bigger. They provide RNA and other materials to the oocyte, which allows it to develop faster
Describe the experiment that showed the importance of maternal mRNA.
Shows that maternal mRNA is necessary for early development, even if embyronic mRNA transcription is not.
1. Take a sea urchin zygote and shake it to separate into two cells, one with a nucleus and one without
2. enucleated cell will still partially develop (it will reach blastula stage)
*Note: protein synthesis is necessary still in all stages of development. Mammals need embryonic mRNA synthesis even in early stages.
How to make so much maternal mRNA without nurse cells?
You just make it for a long time
How is maternal mRNA so stable in oocytes?
specific proteins bind the 3' UTR
What keeps the maternal mRNA from being translated right away in eggs?
specific proteins bind the polyA tail
How are specific mRNAs localized?
microtubules transport mRNAs throughout the cell
when genes inherited from mother and father are differentially expressed in embryo
What affects can imprinting have on the developing embryo?
gynogenetic embryos will reach a stage of arrested development, with a small placenta
androgenetic embryos will result in retarded embryo growth
Describe the process of fertilization.
1. sperm reaches the outer covering and releases acrosomes
2. The penetration of outer covering triggers Ca++ exocytosis of the acrosomes
3. the sperm fuses with with the egg (membranes) and inserts pronucleus
4. Blocks to polyspermy arise
5. pronuclei fuse and the meiosis is completed.
Describe the fast block to polyspermy.
Happens in some species, like frogs and sea urchins. A depolarization of the egg membrane (~1 second after sperm entry). Transient block.
Describe the slow block to polyspermy.
Happens in most species. Causes a rise in intracellular Ca++, which causes exocytosis of cortical granules and polysaccharides (~1 minute after sperm entry). Cortical granules create a hard covering over the egg, and the polysaccharides absorb water and create a jelly layer between egg and cortical layer.
How do we know that Ca++ controls the cortical granule release and activation of the egg?
If you insert Ca++ ionophores, it triggers egg activation and cortical granule release. On the other hand, if you add a Ca-chelator, the activation is inhibited.
What does activation of the egg lead to?
Increased protein synthesis from maternal mRNA, increased RNA synthesis, increased metabolism, and rapid DNA synthesis and cleavage.
Why does cleavage in the early embryo not lead to a greater size?
Normally the cell cycle has a growth stage, but in early development these stages (G1 and G2) are skipped, which means cytoplasm is not created and a lot of time is saved.
Explain how cyclin dependent kinases control cell division.
CDK complexes with a cyclin
MPF (m-phase promoting factor, a protein) is made because of this, activating the cell cycle.
When cyclin is degraded, the MPF falls apart, and cell cycle ends.
Describe the role of the cytoskeleton in cell division.
Microtubules separate sister chromatids. Microfilaments lead to cleavage furrow forming.
Describe the role of the centrosomes in cell division.
The pattern of cleavage depends on the position of the centrosomes.
What factors does the cleavage pattern depend on?
Positions of the centrosomes and the yolk.
What determines whether mammalian embryonic cells are part of the trophoectoderm or the inner cell mass?
The position of the cells in the blastocyst determine whether they'll develop into trophoectoderm or inner cell mass. The position of the cells is determined by compaction during cell division (which means that the level of cell-cell interaction is increased).
What is the MZT?
The point where maternal mRNA is eliminated and embryonic mRNA transcription is activated. At this point, the rapid synchronous divisions stop and slower, asynchronous divisions happen.
What controls the MZT?
Determined by the ratio of nuclear DNA to cytoplasm.
List the different levels where gene expression can be controlled.
Chromatin modification, transcription, mRNA processing (post-transcription), transport to cytoplasm, translation, post-translational modification.
How can you modify chromatin to regulate gene expression?
Methylate DNA or acetylate histones. Modification to histones can be passed along cell division and be propagated along chromosomes.
Describe the importance of global chromatin modification.
During fertilization, the egg is activated by the global demethylation of the nucleus (except for those genes that are imprinted).
How do transcription factors regulate gene expression?
Combination of regulatory proteins, some that activate and some that repress genes. The concentrations and sum of the transcription factors lead to the level of expression of genes.
Describe some ways of maintaining patterns of gene expression.
positive or negative feedback
What are some post-transcriptional controls of gene expression?
Proteins that bind to the 3' UTR can block translation.
What are miRNAs?
Micro RNAs. They are small non-coding pieces of RNA that that are complementary to the 3' UTR of mRNA and will block translation or promote degradation.
What are some techniques to study gene expression?
Genetic screen, microarray analysis, protein staining, in situ hybridization, genetic editing, RNA sequencing, direct injections of RNA (where it's not normally found).
What are siRNAs and morpholinos?
Small interfering RNAs that lead to degradation of target RNAs that
What are genetic screens?
Mess with genetics (over- or under-expression) to see the results
How do you make a transgenic organism and why are they useful?
Insert a foreign gene into an organism.
How do you gene edit?
What are the advantages and drawbacks of gene editing?
Pro: can be applied to just about any organism
Con: the off-target effects of guide RNA
Why do germ cells arise so early?
- They need to maintain a huge amount of pluripotency
- They also need to set aside cytoplasmic determinants
Give an example of a cytoplasmic determinant.
- P granules in nematodes
- bicoid in Drosophila
Describe some key features of cytoplasmic determinants
- mRNA or proteins present in egg before fertilization
- unevenly distributed to daughter cells to drive differentiation
Describe potential consequences of cleavage
- reduction of cytoplasm/nucleus ratio
- generate different cell environments and interactions
- segregation of determinants into different cells
What are required for germ cell determinants in Drosophila?
Maternal determinants (the germ cells develop in the posterior end). This makes sense, because germ cells need to pass on maternal determinants to progeny.
Describe an experiment to show evidence for germ cell determinants.
In an embryo, use UV radiation to ablate the cytoplasm at the vegetal/posterior end that contains polar granules. You'll see that the organism can grow up normally but becomes sterile.
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