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241 Cards in this Set
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LECTURE 23
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Lecture 23
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what is the signal that the cell receives?
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Ligand
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What receives this signal?
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Cell surface receptors
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The ligand activates a ? in the ? and what does it do.
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allosteric change; alters the shape and therefore function of the Intercellular domain
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An allosteric change is an example of ? signaling
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Direct
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Indirect signaling is done through?
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signal transduction pathways
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When the ligand binds the cell sueface receptor, what happens?
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It brings together two receptors
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When these two receptors are brought together, what hapens?
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Their kinases on their ends phosphorlates sequences in the other receptor
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What happens to these phosphorlated sites?
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they are recognized by STAT proteins
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What happens to these STAT proteins?
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They are phosphorlated by the kinase causing them to dimer (come together)
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What does this newly formed STAT dimer do next?
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moves into the nucleus and binds specific DNA sites and starts transcription.
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What binds the cell surface receptors in this case?
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Signal molecule
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once the signal molecule binds, what happens?
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two receptors are brought together
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When the two recpetors are brought together what do they do?
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Phosphorlate each other other.
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This phosphorlyation of the recpetors does what?
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recruits Grb2
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Grb2 does what
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associates with SOS
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the association with SOS helps Grb2 to..
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attach to the Ras kinase
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How do we activate the Ras kinase
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GDP on the Ras is changed to GTP
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Once activated, what does the Ras kinase do?
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activates the MAPk pathway
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What does the MAPk pathway do then?
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phosphorlates transcription factors (Jun)
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Where do these transcription factors go and what do they do?
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into the nucleus and activate transcription
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Eukaryotes are not activated by ? and require ?
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Direct ligand to regulator contact; communication of the ligand through signal transduction pathways
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Three ways signaling helps modify transcription
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-Unmasking an activating region
-Transport into and out of the nucleus (STAT) - Brings together activator/repressor pieces |
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first way unmasking and activating region is done
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a change in the DNA bound activator, revelaing a perviously buried activating region
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Second way
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release of a protein that was bound, blocking the activating region
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how Transport into and out of the nucleus signals transcription
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When not active, regulators are found in the cytoplasm. The signaling ligand causes them to move into the nucleus where they regulate transcription
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how activators and receptors some times come in pairs activates transcription
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in the absence of its ligand glucocorticord receptor (GR) is found in the cytoplasm. when its ligand is bound it moves into the nucleus and binds GRE sites
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What does transcriptional scilencing mean?
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that the silencing is a position effect
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What does the position effect mean?
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a gene is silienced because of where it is located, not in response to a specific signal
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What does it mean to say the repression of gene expression can be global?
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OVer very long stretches of DNA
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Most common form of silencing is associated with ?
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Heterochromatin (denser)
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where is heterchromatin usually found?
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in the telomeres/centromeres
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what happens if a gene is moed into the telomeres or centromeres?
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it is turned off
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another way transcription can be silienced?
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Methylation
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what does DNA methylation do?
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in hibits the binding of proteins. including transcriptional machinery and therefore blocking gene expression
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chromatin at the telomere is ? acetylated
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less. (more methylated)
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Gene silencing in yeast is mediated by ? and ?
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deacetylation and methylation
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First step in this process
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DNA binding protein RAP 1 binds the telomere
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Then Rap1 recruites ?
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SIR proteins
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How many proteins make up the Sir?
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three
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What does the Sir2 protein do?
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It is the histone deacetylase enzyme and it begins to deacetylate nearby nucleosomes
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once Sir2 has deacetylated the nucleosomes what happens?
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the unacetylated tails then bind Sir 3 and Sir 4
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What do Sir3 and Sir 4 do?
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They recruit more Sir complex
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When does methylation stop?
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When there is no more Rap1 protein
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What is the stop signal for Rap1
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histone methlyation
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When a gene can be leaky and not completely turned off, what happens?
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The DNA is methlyated
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after the DNA is methlyated, what happens?
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Proteins bind the methlyated groups
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What do these bound proteins do next?
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They recruit Histone deacytalase and chromatin remodeling proteins
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What does these histone deacetylase and chromatin remodeling proteins do?
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bind the DNA structure and condense it to make it super tight (heterochromatin)
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Impritting is...
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When out of two alleles, only one is expressed.
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If the chromosome is unmethlyated...
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its insulator site will bind CTCF
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when this happens, ? is turned on, and ? is turned off
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H19= on
Igf2=off |
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If the chromosome is methlyated...
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its insulator site will be blocked
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When this happens ? is turned on, and ? is turned off
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Igf2 = on
H19 = off |
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DNA methlyation is maintained through cell division by...
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Maintenance methlyase
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Maintenance methlyase does what?
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Recognizes the hemi-methlyated cytosin after replication and methlyates it
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This is an example of epigenetics and it shows that gene expression can be inherited across ...
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generations, not just a single cell division
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LECTURE 24
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Lecture 24
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Three ways cells express genes in development
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-mRNA localization
- Cell to cell contact - diffusion of secreted signaling molecules |
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How does mRNA localization help to distinguish between identical cells?
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by asymmetrically localizing mRNA
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Where does this process happen?
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In the cytoplasm
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How do the mRNA locoalize?
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By hitching a ride on actin filaments
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How does it hitch a ride?
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an adaptor protein binds the UTR of the mRNA
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Then what binds the adaptor protein
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The myosin motor protein
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What do these motor proteins do?
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They walk along the polarized actin filament and bring the mRNA with them
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Which direction in the complex moving?
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Towards the + end
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CASE STUDY FOR mRNA localization
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mother and daughter cells swtiching mating types
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A ? cell can switch mating types, but a ? cell cannot
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Mother
Daughterq\ |
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Mating type swtiching is controlled by what protein?
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HO
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If the HO is expressed?
If the HO is not expressed? |
It can switch mating types
I cannot switch mating types |
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What represses the HO protein?
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Ash1
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What do the mother cells do to the ash1 and what effect does it have>
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The mother cells transcribe ash1 and it is localized to the daughter cell so then the HO protein is repressed in the daughter cell
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A cell can influence which genes are expressed in neighboring cells by
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Cell to cell contact
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How do these cells come in contact?
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Through ligands and receptors
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when the ligand and receptors contect, what happens next>
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it makes a kinase
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What does the kinase then do?
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causes the phosphorlyation of DNA bind proteins in the nucleus
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What does this phosphorlation cause the DNA binding proteins to do?
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activate/repress transcription for certain genes
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What happens to the receptor?
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It is cleaved and the intracytoplasmic domain moves into the nucleus
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What can this cleaved domain do?
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Regulate transcription of sets of genes
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CASE STUDY: Cell to cell contact
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nerve-skin cell formation
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Developing nuerons produce a cell surface signaling molecule called
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Delta
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What does delta do?
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binds its receptor Notch
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What happens to the Notch?
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Its intracytoplasmic portion is cleaved and goes into the nucleus
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What does the Notch do inside the nucleus?
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Binds with Su(H)
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What does the Notch-Su(H) complex do?
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they activate the expression of neuronal gene
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What does this lead to?
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It makes the nueron cells become epidermal cells
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Diffusion of a secreted signaling molecule ..
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Helps decide where what will form in the cell
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Cells located at the front (anterior) portion of the embryo will form..
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Portions of the head
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how is positional information distributed?
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by the extracellular gradient
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Where do these signaling molecules come from?
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The morphogen source
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The closer a cell is to the morphogen source...
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the more signaling molecules it recieves
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the more signaling molecules it recieves...
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the more activated surface receptors it has
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more activated receptors...
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More genes expressed
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LECTURE 25
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LECTURE 25
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Once an egg is fertilized, the nucleus of the embryo undergoes
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Multiple rounds of mitosis without cell division
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The bag of cytoplasm with many nuclei inside is called the
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syncytium
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how rounds of nuclear divison does the cell go through?
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14
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What happens after the 14th round
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the nuclei migrate to the outter edge of the cell and start to form cell membranes around the nucleus
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Cells in the posterior region form
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pole cells
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How is the dorsal-ventral patterning in the early drosphilla controlled?
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by a protein called Dorsal
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How is Dorsal initially distributed in the cell?
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evenly
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When can Dorsal enter the cell?
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Once the nuelci have made their way to the outter portion of the cell
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How is the transportation of Dorsal into the nucleus monitored?
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Spatzle
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Where on the cell is the Dorsal allowed to move into the nucleus?
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The ventral region (bottom)
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Where is spatzle most concentrated?
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Ventral region
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Once the cell is fertilized
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Spatzle binds toll receptors
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After binding and activating the toll receptors
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the toll receptors activates Tube
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Tube activates
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Pelle
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Pelle does what
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It destroys the cattus protein on the Dorsal
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attacking the cactus protein by Pelle does wat
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Allows Dorsal to move into the nucleus
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The three genes ? ? ? are turned on by ?
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Twist
Rhomboid Sog Dorsal |
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Twist is turned on only when concentrations of Dorsal are
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High (low affinity)
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Rhomboid is turned on when concentrations of Dorsal are
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moderate-to-high
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sog is turned on when concentrations of dorsal are
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low (high affinity)
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Highest dorsal leverl
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mesoderm
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medium dorsal level
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Ventral NE
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low dorsal levels
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dorsal NE
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What does Snail do?
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prevents the expression of rhombiod and sog in the mesoderm
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snail is only expressed in the
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mesoderm
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how does snail stop the expression of rhomboid and sog?
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it binds the dorsal binding site for rhomboid and sog in the mesoderm and therefore prevents dorsal from turning on expression of these genes
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Therefore rhomboid and sog are only expressed
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in lateral regions that formthe neurogenic ectoderm (NE)
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At fertilization the drosphilla egg contains two localized mRNA called
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bicoid mRNA
oscar mRNA |
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the bicoid mRNA is found
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at the anterior pole
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the oscar mRNA is found
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at the posteior pole
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the oscar mRNA encodes for a RNA binding protein that
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assembles polar grandules
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these polar grandules are responsible for
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the development of the posterior embryp
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Where is the oscar mRNA first deposited?
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in the anterior region
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This anterior region is called
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oocyte
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when the oocyte starts to get bigger
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the oscar mRNA get transported to the posterior end
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How does this relocation occur?
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the oocyte is very polarized so when it goes, microtubles from the nucleus extend to the + end and the oscar mRNA interact with the + end of the microtubles
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The cells that inherit these oskar mRNA and become fertilized form
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pole cells
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these pole cells help to
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develop the posterior side of the embryo
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the UTR of the bicoid and oskar mRNA play a crucial role by
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moving the mRNA to where they need to be
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If the UTR of a bicoid mRNA was switched to an oskar mRNA
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the oscar mRNA would stay in the anterior end and cause formation of pole cells in abnormal places
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LECTURE 26
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LECTURE 26
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Bicoid regulatory proteins diffuse how
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from anterior to posterior
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this diffusion forms a
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bicoid protein gradient
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Does this bicoid gradient need to utilize cell surface receptor? why?
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no because it diffuses freely
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High levels of bicoid are required where
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in the anterior region to produce structure like the head
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high levels of bicoid control expression of
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Orthodenticle gene (low affinity)
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High/Medium levels of bicoid activate
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Hunchback gene (high affinity)
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The hunchback gene is required for development of the
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thorax
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Hunchback is transcribed from two different promotors
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-one activated by the bicoid gradient
- one in the developing oocyte |
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When hunchback is activated by the developing oocyte
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RNA is distributed evenly
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What and where are Nanos proteins
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Nanos proteins bind hunchback mRNA in the posterior region
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What does this do?
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Inhibits translation of the Hunchback mRNA in the posterior region
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What forms because of this?
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a steep Hunchback gradient
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Hunchback protein is a
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repressor
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Since it is a repressor, it limits the expression of
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gap genes
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the three gap genes are
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Kruppel
knirps giant |
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High levels of Hunback repress
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Kruppel (low affinity)
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High/Medium levels of Hunchback repress
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knirps (moderate affinity)
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Low levels of hunchback repress
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giant (high affinity)
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gap genes are
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repressors
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what genes do these gap genes repress?
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pair rule genes
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pair rule genes are also called
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even-skipped genes (eve)
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eve is expressed in
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a series of alternating stripes along the embryo
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each eve stripe is controlled by
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an enhancer
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ASK FOR HELP ON THE EVE STRIPE QUESTIONS
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ask for help eve stripe questions
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LECTURE 27
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LECTURE 27
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3 ways GENE EXPRESSION can be altered during evolution
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- a gene that helps determine body patterning can be expressed in different patterns
- a gene that helps determine body can be altered so it develops new functions - Target genes of a specific patterning gene can acquire new DNA regulatory system |
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Pax6 is..
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a gene that yields for normal eye development
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Completely remove Pax6 ...
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No eyes
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Misexpress Pax6 in cells that don;t normally express it,,
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Get development of eyes where they aren't usually expressed
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Antp is ..
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patterning gene that controls the development of the mesothorax (LEGS)
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the mesothorax develops
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midlegs and wings
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the metathorax develops
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hindlegs and halteres
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Ubx is the gene that controls..
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development of of the metathorax (HALTERES)
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The Ubx represses
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Antp in the metathorax
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Ubx allows expression of
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hindlegs and halteres
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Mutation in the Ubx allows..
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Antp to be expressed in the metathorax, so we get midlegs and wings in the metathorax
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Mutation in the Ubx regulatory sequence can cause..
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Ubx expression in the mesothorax, so we get hindlegs and halteres in the mesothorax
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Pax6 expresses
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eyes
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Antp expresses
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Wings and midlegs
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Ubx expresses
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Hindlegs and halteres
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LECTURE 28
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LECTURE 28
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RNAi stands for
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RNA interface
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What does RNAi do
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silences genes using small regulatory RNA
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3 ways RNAi silences genes
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-trigger the destruction of an mRNA
- Blocking translation of an mRNA -Inducing chromatin modifications of a target gene |
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RNAi is found in
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all eukaryotes
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the two important types of small regulatory RNA
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siRNA
miRNA |
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how is siRNA produced?
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produced in the lab from dsRNA
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how is miRNA produced?
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from a precursor mRNA expressed in the cell
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What is this precursor mRNA called?
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primary RNA of pri-RNA
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What is inside this pri-mRNA
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hairpin like pre-mRNA
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where can these pre-mRNAs appear
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anywhere. coding or noncoding regions
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Once pre-mRNA hairpins have formed on pri-mRNAs, what happens
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the pre-mRNAs are cleaved from the pri-mRNA
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what does this cleaving?
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Drosha and Pasha
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once the pri-mRNA is cleaved by Drosha and Pasha, what happens next
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it is exported to the cytom plasm
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Once the pre-mRNA is in the cytoplasm, what happens?
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its terminal (loop) end is cleaved
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what does the cleaving, and what is the outcome?
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Dicer. it creast a 22 bp dsRNA
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this newly formed dsRNA is called
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miRNA
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Once siRNA is introduced to the cytoplasm, what happens?
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Dicer cleaves the dsRNA
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What is the result of this cleavage on the newly introduced dsRNA?
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siRNA is formed
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What allows Dicer to recognize and bind RNA?
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its PAZ domain
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What allows Dicer to cleave the RNA?
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RNAse III domains
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Once siRNA and miRNA are formed, what happens
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They are unwound to produce two ssRNA
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What are these two ssRNAs called and which one is important
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Guide and Passenger
Guide |
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What is the job of the guide RNA?
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to lead the RISC complex to the target mRNA
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What is the central component of the RISC complex
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Argonaute
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what does Argonaute do
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it slices the ssRNA-mRNA hybrid in the middle of the quide (ssRNA)
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What is Argonaute also called
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Slicer
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what does Argonaute have that allows it recognize and bind RNA
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PAZ domain
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How can we use siRNA to knock down genes?
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introduce dsRNA into the cell that is complementary to a gene of interest
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How do we monitor if the gene expression has been knocked down?
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Immunoblotting
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What is the first step to immunoblotting
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all the protein is extracted from the cell
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Once the protein is extracted, what happens?
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it is denatured
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What does this denaturng and how?
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SDS and BME. reduces the disulfide bonds within proteins
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What is the result of treating the proteins with SDS
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it gives them a negative charge
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Once these proteins have a negative charge, what happens?
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They are ran on electrophoresis to separate them based on size. (smaller ones go further)
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What allows us to see the proteins on the gel?
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treating them with coomassie blue
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What happens next
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the proteins are transferred to a membrane
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once the proteins are on the membrane what happens
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the are treated with antibodies
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what do they antibodies do
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bind specific proteins
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after the antibodies have bound a specific proteins what happens
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a chemical is added that is processed by an enzyme in the antibody that produces a color
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LECTURE 29
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LECTURE 29
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What does it mean that the RNAi pathway is very efficient
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only very small amounts of dsRNA are needed to induce a shutdown of target genes
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Two reason why it is so efficient
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-RNA-dependent RNA polymerase (RdRP) makes more dsRNA off of the RNA template
- RISC complex can cleave several mRNAs |
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How can small RNAs silence genes?
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Condensing the chromatin (low acetalyation, high methlyation
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Since centromeres are usually silenced, they have
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low histone acetylation and high methlyation
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The loss of dicer or Argonaute leads to the moss of
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methlyation and the centromeres are no longer silenced
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When centromeric DNA get transcribed what is produced
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dsRNA
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where does this dsRNA come from
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centromere repeats
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Once the dsRNA has been formed, what happens
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Dicer cuts the dsRNA into small RNA
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What does this new small RNA do
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Bind the RITS complex
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what does this small RNA-RITS complex do next
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binds the transcripts coming from the centromeric repeats
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What does the binding of the small RNA-RITS complex to the transcripts coming from the centromeric repeats do
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recruits histone modifiers Swi6 and Clr4
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what do the histone modifiers Swi6 and Clr4 do
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they methlyate and silence the chromatin
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Lin-14 is responsible for
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stage 1 larval development
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What does the worm need to process to stage 2 larval development
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Lin-14 needs to be absent
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How does this happen
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Lin-4 target Lin-14 and decreases the levels of Lin-14
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When does Lin-4 start to rise
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stage 1
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and what does it do
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targets Lin-14 and decreases its levels
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Lin-4 is responsible for
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Stage 2 larval development
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Between Lin-4 and Lin-14, which one is the repressor
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Lin-4
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what does Lin-4 encode for
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small RNAs
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How does Lin-4 repress Lin-14
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it binds its Lin-14 3' UTR site
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how Lin-4 has bound Lin-14 3' UTR site
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with imperfect complementarity
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what does this mean
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it doesn't have to be an exact match
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The better Lin-4 can Lin-14 means that
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it has more binding sites
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The more binding site,
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the greater transcriptional repression of Lin-14
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