Chp 1&2: Development Flashcards
(104 cards)
0
Q
Cleavage
A
- process of creating blastomere (smaller and smaller cells due to division) ->blastula
- leads to a cavity in all organisms called the blastocoel/blastopore
1
Q
Fertilization
A
Brings together 2 genomes
Ex: frogs have chemical signal that tells them to produce gametes
Females->produce yolk->zygote
2
Q
Gastrulation
A
-gastrula
-germ layer formation (3 layers):
Endoderm: lining of intestines/lungs
Ectoderm: skin, nervous system
Mesoderm: skeletal, muscles, parts of organs, blood, becomes gametes
-moving of the blastomeres resulting in the germ layers
3
Q
Organogenesis
A
-interactions, rearrangements, migrations
Ex: notochord= works as a signal for different tissues -> nervula
4
Q
Metamorphosis
A
- larval stage (non sexual) to sexually competent adult
- different depending on group of organism
5
Q
Germ cell - Gametogenesis
A
-tends to be isolated during development due to the different signals
6
Q
Development
A
Aristotle 350 BC- used chickens
Limited to multicellular except in yeast cells
Embryology= old name
Continues past gestation (humans late 20s)
7
Q
3 approaches to dev bio
A
Anatomical
Experimental
Genetic
8
Q
Anatomical
A
Blastomeres-cells & fate
Comparative embryology - dev diff
Evolution
Teratology- teratogens: result in birth defects
-observe deformities to inform what went wrong
Mathematical- pos. & neg. feedback
-chemical
9
Q
Ovoviviparity
A
Eggs w/ yolk
Hatch internally
10
Q
Oviparity
A
Egg layers: birds, frogs, insects, monotremes
11
Q
Viviparity
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Placental mammals
12
Q
After Aristotle …
A
Nothing happens bc of religion
- William Harvey (1651)
- late 1600s: Enlightenment->science kicks in
13
Q
William Harvey
A
“All animals are from eggs”
NO spontaneous generation
Tried to find the mammalian egg
-used deer
14
Q
1672: Marcello Malpighi
A
Microscopes
-microscopic accounts of chick
How dev. occurs?
-epigenesis -> organs from scratch vs. preformation -> everything is already there, it’s just miniature
-no cell theory, so no limits on how small something was
15
Q
Kasper Wolff
A
Supports epigenesis
Watches late tissue formation
16
Q
1820s: several German scientists
A
Germ layers
Microscopes get perfected
-new staining techniques that allowed them to see small structures
*ectoderm, endoderm, mesoderm
-interaction was critical
~of the layers: to know what they’re purpose is
-relationship among early embryos and their structures across species
-the closer related… The longer it takes to distinguish embryos (humans + chimps)
-as dev progresses, characters go from generic -> specific
17
Q
2 kinds of cells
A
Epithelial - sheets
Mesenchymal - wanderers
18
Q
Morphogenesis
A
Due to a limited amount of cell activities of cell activities
*where they go + how much they divide can have cell shape change
Mesenchymal-> epithelial-> tube-> sheet
19
Q
Fate mapping
A
-almost the point where medicine comes in
-end up with cell lineages
Each cell is a daughter
-tunicates
Look like tadpoles as larvae -> bag of goo
Cytoplasm had diff colors BC of germ layers
Test fates by removal of mesodermal cells
20
Q
Genetic Labeling
A
1920: Hilde Mangold + Hans
Chimeras
21
Q
Chimeras
A
2 genetically diff species mixed
-1st ones done on newts
-chick + quail: easily identified cells
>Condensed dna
>have specific antigens
>neural crest cells
22
Q
Transgenic chimera
A
Today: put in green fluorescent + protein (GFP)
23
Q
Life Cycles (review)
A
-Fertilization>hatching=embryogenesis 1 cleavage 2 gastrulation 3 organogenesis 4 gametogenesis 5 metamorphosis
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Cleavage
Rapid mitoic division
Zygote> blastula
*volume stays the same
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Gastrulation
Cell movement resulting in germ layers
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Organogenesis
- formation of organs
- a lot of cell communication
- some organs = multiple germ layers
- cells migrate
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Metamorphosis
-maturity> related to gametogenesis ("not ready") where germ cells are set aside for reproduction + protected
*fated of cells depends on what they're next to
>all other cells are somatic (46 chromosomes, go towards creating the body)
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Frog Life Cycle
-when conditions are good (enough sun, good temps) females will make yolk in the liver which is packed into the eggs
-Fertilization
>germ cells move into the gonads from a hiding spot in the endoderm
-eggs come from oognia
-sperm comes from spermatogonia
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Mitosis (in frog)
Germ cells from oognia or spermatogonia
Primary oocyte spermatocyte> meiosis(2n->1n) > homologous chromosome pairing *shuffling* daughters = haploid> 1n - secondary oocytes
-in the egg meiosis stops early + sits in stage of haploid
Until it reaches sperm...egg complete
-nucleus in the egg = "pronucleus"
-nucleus + pronucleus combine to form zygote (2n)
-as sperm hits egg> cytoplasmic rearrangement (change of color, movement)
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Cleavage (in frog)
- volume remains the same
- blastula= 10s of thousands of cells
- animal pole divides quickly (sm. cells)
- vegetal pole is slow (large cells)
- blastocoele forms (fluid cavity)> animal pole
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Gastrulation (in frogs)
- sperm entry defines dorsal surface of organism
- as cells migrate in through blastopore (lip, gray crescent) they become mesoderm
- the cells outside > ectoderm
- blastopore becomes neural groove eventually
- the large yolk-filled cells in the vegetal pole> become endoderm
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Organogenesis (in frogs)
Mesoderm in the most dorsal
-neural groove
-neural tube> nervous system
Ectoderm above notochord becomes tissue in your spine
Embryo> neurula
Ectoderm grows over the neural tube (birth defect if doesn't grow)
Mouth & anus form
Muscles form (dev point when start moving, where it hatches)
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Cell specification
"Presumptive tissue"
1) specification: if put in a neural environment, no signals> follows fate
2) determination: follow fate regardless of environment, irreversible
Ex: tunicate- in the cleavage stage, the 1st blastomeres determine fate
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Types of specification
1) autonomous >specific blastomeres translates to specific tissues/parts determined by cytoplasmic constituent
*proteins- transcription factors
*mRNA
>fates are invariant
>most invertebrates
2) conditional specificity
-all vertebrates a few invertebrates
-fate is determined by your "friends"(environment)> happens a little later
-little invariant fate assignment
-can frequently switch fates (good thing)
-cell rearrangement> migrations> specificity: *development gets regulated which allows cells to acquire a variety of characters
3) syncytial specificity
-mostly insects (all insects do this)
-localization of cytoplasmic constituants
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Genetics
-visible mutations on the chromosome lead to the nucleus being determined as holding the info/DNA (variable)
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Cell Fates & Cloning
-do cells become differentiated?
-easy in the 1950s (thought of in 1890s)
>remove nucleus from oocyte, remove the donor nucleus from cell, transplant donor into oocyte
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Somatic nuclear transfer (cloning)
1956: King + Briggs
-used tail bud nuclei for a SNT
>result: nothing
-used germ cells nuclei
>result: frog
*establishes that the DNA is being modified/responsible
DNAs fate= determined
Clones= twins
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1975: serial Transplantation
Took adult frog foot web cells
>ectoderm (skin, nervous tissue)
>very specialized
-do the SNT>embryos>gastrulation
-go into blastula (pregastrulation)>take nucleus + transplant into another oocyte which survives up until tadpole stage then dies
*proved reversible differentiation is potential in all cells
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1997: Dolly (sheep)
-G1 stage: mammary cells (diff breed) from one sheep
-enucleated an oocyte (in 2nd meiotic phase)
-fused the cells w/ electroshock (used to get the egg cell to believe it was fertilized) in this case>membrane fusion
-did process with 343 cells
>result: 1 Dolly
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Totipotent
Creat every cell possible
- no genes have been lost/mutated during differentiation
- phenotype is not identical
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How do you turn on/off genes?
1) every cell w/ a nucleus (RBC don't have nucleus) is the same DNA
2) unused genes are not destroyed so potential for expression exists even if everything's shut off
3) only a small % of the genome is expressed in a cell
>RNA should be specific
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Fruit fly gene expression
-used polytome chromosomes
-DNA replication but no mitosis 2^9 vs. 512
-150 times the normal amount
-found that regions of the chromosome
>"puff out" when transcribed
-DNA rep. found in salivary glands
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Other gene expression studies
- confirmed point 3> DNA RNA hybrids
| - label DNA or RNA seq of interest and put them in w/ RNA isolated from cell
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Diff. Gene Expression Techniques
| RT PCR
```
RT= reverse transcriptase (virus)
PCR= polymerase chain rxn
```
* take RNA + turn that into DNA using RNA virus (RT)
* in the PCR, polymerase is used
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Diff gene expression technique
| cDNA
Complementary DNA sequence bound to a plate
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Insitu hybridization
- labeled anti sense RNA (radioactive or dyed)
- look for areas of expression
- can look at quantity of expression
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Transgenics
-allows you to look at gene function during normal dev
-doesn't damage dev of organisms
-can inset cloned DNA into cells
>microinjection
>transfection -low intake
>electroporation -speed intake by shocking EX: sodium shock
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Transposable Elements
```
"I am legend" movie
-retroviral vectors (Gene therapy)
>near 100% insertion (you'll get results)
>replace viral proteins w/ phyload
-marker product (GFP)
```
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Chimeras (mice)
- take a blastomere from mouse A + put it in mouse B early, it contributes to all tissues
- took from inner cell mass then they were totpotent
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Gene Targeting
- from knockouts
- if they have 1 good copy then the organism will develop
- if knockouts are crossed then you'll get heterozygotes
* break gene l, microinject, then cells swap out bad gene for good gene
* Bone morpho protein/BMP 7 Knockout: the heterozygote develops normally + the knockout has no eyes or kidneys
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Newer: Antisense RNA
-inject a large amount of it into cell which makes the cell destroy both sense and antisense forms of RNA
>cell does all the work
-Morpholino Antisense (permanent poison)
>hard for the cell to destroy RNAs
>double stranded RNA triggers an antiviral response
52
Differential Gene Expression
~25,000 protein encoding genes
-dev genetics
>how do you take same genes + create diff cells/tissues
>how do you go from genotype -> phenotype
*diff from cell or molecular bio is the level of interest
>want to find out how the cellular processes function on a tissue/cell level
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Gene Expression
-proteins lead to everything else except ribosomes
-occurs at multiple levels:
1) Diff Expression -> RNA
2) Selective nRNA processing
-which nRNA->mRNA goes to cytoplasm
-intron + exon splicing (antibodies)
3) Selective Translation
>RNA to protein
4) Differential Protein Modification
>degradation
*diff signals to express here AND for how long*
>activation
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Chromatin (active or repressed)
>complex of DNA + proteins (histones)
-nucleosome= basic unit of chromatin-> DNA (2loops) wrapped in 8 histones
~140 base pairs
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Chromatin
String of nucleosomes which are connected by H1 linker proteins (histones)
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Histone
Cause nucleosomes to get wound tight
| -organize DNA strands into nucleosomes by forming molecular complexes around which the DNA winds.
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H1 confirmation represses transcription
(take yarn off ball when knitting)
| -when H1 linkers are removed, it allows transcription factors to get in there
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Nucleosomes and H1
Limit access to DNA
-default condition = repressed
-specification/diff. -> cell/tissue specific change to the repressed state
> use histone acetyltransferase(can add acetyl groups)
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Methyl + acetyl groups
Location: on tails
- tighten or loosen nucleosome
- remove methyl= loosen
- remove acetyl= tighten
- histone methyltransferase adds methyl groups (tightens)
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Introns + Exons
```
Introns= loose
Exons= exits nucleus/keep
```
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What is "the gene"?
*promoter: polymerase binding/initiation
>upstream (5')
*transcription + translation termination helps form poly-A tail
-before leaving nucleus:
>nRNA will get a 5' cap of methylated guanine
>reverse polarity so there's no 5' end
*poly-A tail
>needed to bond to ribosome
*3' poly-A tail
>protects from exonucleus
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Promoters & Enhancers
-determine when + where genes are expressed
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Promoter
Upstream- bind polymerases and also transcription factors
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Basal transcription factors (TFs)
Necessary for polymerase binding
| Ex TATA binding protein (TBP)
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TFIID
Fraction of a cellulose extraction
- foundation of the complex of proteins needed for initiation
- stops nucleosome formation
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TFIIA
(Larger protein)
- binds w/ TFIIB so that RNA polymerase can bind
- factors E, F, H release RNA polymerase + unwind helix (like helicase)
- regulated by TBP Associated factors
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TBP Associated Factors (TAF)
Can help modulate activity of the RNA polymerase activity-bound upstream of promoter + is tissue specific
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Enhancer
Control efficiency and rate for a specific promoter
*importnat in tissue specific exp.
-can be 1000s of base pairs away but on the same chromosome (limit)
-5' (upstream) or 3' (downstream)
-function= combine w/ transcription factors
-nothing can bind to it
-removing methyl groups
-most genes require enhancer activity
-multiple enhancers + multiple TF can all work on 1 gene
>want to have enhancer to work the right way + same place
-enhancers can also inhibit
-mixed activity
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Transcription Factors (TF)
-proteins that bind to enhancers (DNA seq) and promoters
-activate or repress (variably) a lot or little
-grouped into families
>small amino acid changes, change binding
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Hox Genes
-codes for TF
-axis formation
Pou= pituitary and neural fates
Lim= head
Pax= neural and eye dev
-basic structure= combination of Leucine zippers, Helia loophelia, zinc finger
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3 TF domains
1) DNA binding domain
2) Transactivating domain
>important in activating/suppressing transcription
>binds to proteins (TFIIS modifying histones)
3) protein-protein interaction domain
>other TFs or TAFs(transcription associated proteins) can come in and modify activity
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Ex 1 of TF domains
| MITF
-TF that's active in ears and pigment cells
-heterozygote (1 functioning form of MITF)
>deaf, multi colored eyes, white forelock
>helix loop helix structure
*protein-protein domain allows it to dimerize w/ MITF
*can then bind to DNA
*transactivating domain binds w/ a TAF (acetyl transferase)
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Ex 2 of TF domains
| Pax 6
- active in mammalian eye, nervous system + pancreas
- contains 2 DNA binding domains
- pax 6 binding seq. found in lens gene enhancers and endocrine cells in pancreas that are important in insulin/glucagon (Diabetes)
- can activate or suppress
- pax 6 + sox 2 have to be present together for lens dev (eyes won't form)
- sox 2 is only present in ectoderm that's been exposed to optic vesicle
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3rd Binding site of Pax 6 TF domain
- repression or activator
- critical in stopping lens formation outside eye
- when expressed it does a positive feedback (becomes permanent enhancer)
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Transcription Factor Cascades
-TFs are activated by TFs
>Pax 6 is turned on by TF: MBX which is expressed in late gastrulation
-Activation of TFs
>may need a signal to be competent
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Silencers
Repressed that repress transcription
Ex: neural restrictive silencer enhancer (NRSE)
>causes promoter only to be active in neurons
-expressed in all other cells
-works as deacetylase
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Methylation
-DNA methylation
>how expression becomes stable -> adulthood
-promoters can become methylated
CpG seq because cytosine gets methylated but only if followed by guanine
-methylated cytosines stabilizes nucleosomes
>results in TF not binding
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Genes during development
-genes only active in sorry or some active in egg
-methylation of CpG
-imprinting
>disadvantage: lethal alleles exposed
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How does methylation stop transcription?
-interaction w/ histones
-methylated DNA attracts enzymes that further methylated DNA
-methylated DNA stabilizes nucleosomes beyond stabilized methylation of histones
*Mecp2 (protein)> selectively binds methylated DNA and also binds deacytelases
(H1 linker histone) ->attracted to methyl DNA
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Clones
Compare to normal formation -> methylation is messed up
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Normal DNA methylation
maintained during mitosis by DNA methyl transferase (1 strand methylated + the copied strand will be methylated)
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Loss of methylated transferase (mice)
Small shortly after birth, die because of multiple malignant tumors
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Insulators
Seq that binds proteins "insulators" stops activation of adjacent promoters often located between enhancers and promoters
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Dosage compensation
```
Inactivation
Ex: X chromosome
X converted to heterochromatin
Heterochromatin remains condensed
Replicates later than the other chromosome
Forms Barr body
*if not shut down; death because only want same X chromosome shut down in every cell
*early inactivation critic
Shut down isn't always 100%
-15% could still be active (alleles)
-germ s will cause reactivation
```
85
Inactivation
= a chain reaction:
MeCP2 > methylated DNA
Stabilized nucleosomes > deacytelases
EZH2 > methylates DNA & histones
H1 highorder folding (nucleosome)
*all of this initiated by RNA seq that never leaves nucleus
-methylated promoters
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Differential RNA processing
```
(DNA>nRNA>mRNA>proteins)
After transcription> translation
1) mRNA (introns, cap, tail)
2) move from nucleus> cytoplasm
3) translation> post-translation changes
-diff in cells=diff in proteins
-same pool of RNAs but diff sets make it to the ribosomes (called censorship)
-splicing: exons + introns
*combine multiple exons differently
*nRNA contains introns
>pre mRNA
*by processing diff subsets
```
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Example of differential RNA processing
Sea urchin blastula vs gastrula
- more genes are transcribed than expressed
- the stuff not sent out = degraded within nucleus
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Alternate RNA splicing
-normal vertebrate RNA has many introns + exons that get spliced together> shorter seq
-splice sites (introns) decide if it's cut out or left
-spliceosomes= bind to splice sites
>made of combination of RNA + protein (splicing factors)
-diff spliceosomes = diff results
-splice site recognition is 5'
89
Alt. RNA splicing example
| Tropomyasin
-1 gene for Tropomyasin
BUT diff kind (slight modifications) of Tropomyasin in brain, liver, skeletal muscle, etc.
Multiple protein forms
*called: splicing isoforms
90
Alt. RNA splicing example
| BC1-X
1 form inhibits apoptosis
| Other fork induces apoptosis
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Alt. RNA splicing example
| Fruit flies
1 gene in drosophila has 38,016 potential isoforms
| *genome and protenome are not equal
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Differential Splicing
Enhancers etc.->DNA->RNA
-diff proteins recruiting to splice sites
>changing splice activity
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Translation
Controlling protein creation
- mRNA longevity> stabilized RNA leads to more protein
- stability is a function of poly A' tail
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Untranslated region
UTR
(Not turned into proteins)
-determines length
-when experiment alters UTR it increases or decreases the half life of mRNA
-caesin mRNA: half life= 1.1 hours (during lactation= 28 hours)
>protein found in milk
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Oocyte translation
Make and store mRNA that is used much later after fertilization
- necessary for chromatin, membranes, cytoskeleton components
- early cell divisions rely on this
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Bicoid & nano translation
(Insects)
| Localize (mRNA) to specific parts of cell
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Translation regulation
-tends to be negative
-default state= on
-5' cap 3' tail is where this occurs
>important in ribosome binding
*no cap/tail = no translation
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mRNA
Circular
5' held to 3' by proteins *important in unwinding double stranded RNA
3D structure
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Ex of mRNA
| EIF4G
Binds 3'
Binds ribosome
Ribosome binds 5' initiation factor
100
Ex of mRNA
| EIF4G
Binds 5' cap
Interacts with G
So oocytes produce RNAs without cap
-fertilization >capping
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Cytoplasmic localization
3' UTRs
-vegetal localization (yolk rich)
-bicoid (anterior) nanos (posterior)
>if 3' UTR from bicoid and add to RNA it knows where to go
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Post translational
Structural
Enzymes
Phosphokinase
TF
103
For many proteins to become active...
```
Need something else:
-dimer
>MITF - hemoglobin
-cleaved - insulin
-ribosomes - tubules
-ion binding
```
