pastor lecture 2 Flashcards
what happens when screening for temperature sensitve mutants at high temp?
-some cells die at high temp, indicating they are the temp sensitve mutants
how many cells exits in adult vs die durinig development in C elegans?
-959 cells in adult
-131 die during development
What type of cells were not swalowed up?
-the ced (cell death mutants) dont get swalloed up which provides a tool to study the apoptotic pathway
What type of ced is crucial for apoptosis/
ced-3 is crucial for apoptosis to occur
What happens in an egl-1 mutant?
What was occurring?
What saved phenotype?
-egg laying mutant causes eggs to form in the hermaphordite but undergo apoptosis like a male
-apoptosis was occuring when it wasnt supposed to
-ced-3
What ceds initiate cell death?
What caused excess cell death when mutated?
-ced-4 and ced-3
-ced-9, it is a positive regulator cell survival that would inhibit ced-3 and ced-4 activity, but when mutated it causes cell death
What is homologous in humans to ced-9?
-ced-9 mutant mapped to bcl-2 which is an oncogene overexpressed in b-cell lymphoma)
How would you test the expression of human bcl in c.elegans?
What would you introduce, what is the promoter for?
What do you clone?
-put human bcl-2 in c elegans
-introduce gene expression vector which has promoter into c.elegans
-has a promter for HSP16 (turns on if increase in temp)
-clone in cDNA for ced-9 and bcl-2 gene
What happens when hsp-ced-9 construct is in contact with heat shock?
What about hsp-bcl-2
-hsp-ced-9=neurons surve that shouldnt have
-hsp-bcl-2=large aount of worms get extra cells
what is egl-1 like in the apoptosis pathway?
What is analogous to ced-9?
egl-1 is like BAK and BAX which are pro apoptotic proteins
-bcl-2 is analogous
What is ced-4 analogous to, what does this part activate?
What does CASP9 activate?
-ced-4 is analogous to APAF1 which activates procasp9
-proCASP3 leading to CASP3 which is analogous to ced-3 which kills cells
where can apoptotic signals come from?
What stimulates the internal signal?
they can come from internal or extrnal parts of the cell
-TNF which stimulates CASP8 for internal
What does lin14 mutant cause?
What about excess Lin14?
-causes early shutoff of larva stage
-excess causes early larval pathway to be constantly repeated
What are lin14 and lin4?
What does lin4 loss of function cause
-what is purpose of lin 4?
-they are heterochronic mutants (developmental time mutants)
-lin4 loss of function=lin14 gain of function
-lin4 only purpose is to block expression of lin14
-no lin4=too much lin14
What type of lin 14 is always present?
What does lin 4 work as?
the lin 14 transcript is always present
-it works as an RNA since it doesnt code for a protein
how to test what lin 4 codes for?
What does lin 4 bind to?
-run an western blot (RNA test), and implement labeled probes complementary to lin 4
-lin 4 binds to lin14 3’UTR and inibits translation
what is let7 and what does it do?
-it is a heterochronic mutant that keeps dividing as an adult
What is drosha?
what are the 3 things that RISC (RNA induced silencing complex) leads to?
-precursor of microRNA
-leads to mRNA target cleavage
-mRNA deadenylation and decapping
-and translation inhibition
how many bp does miRNA need to be the same as 3’UTR?
What does miRNA recognize?
-they need first 21 bp to be he same
-they recognize complementary miRNA sequence
What is downegulated in lung cancer?
What happens when we downregulate Let7?
-let 7 bc it acts as a tumor suppressor
-causes more Ras protein to form
What are the 3 steps for RNA screening in C.elegans?
what do you clone, and RNAi
PCR from genomic or cDNA template
-clone PCR into vector + transform into cells
-RNAi each gene individually
What does a mutant in the ste of C.elegans cause?
What does mutant of emb cause?
-it can cause sterility so no progeny are formed?
-mutant in emb can causee abnormal growth, the egg will form but the worm wont grow
What does the screening in various cell types look like?
How does the RTK get activated for the pathway of R7?
What happens after the activation, whay is bound and activated after?
Boss binds to RTK which activates it, leading to phosphorylation which is bound by Sh2 domain
-then SH3 binds to proline rich regions activating Ras GEF (sos)
-this leads to active Ras protein which causes downstream signaling
What occurs in the supressor screen approach?
-too much A produced phenotype and then reduction of B eliminates phenotype
What occurs in the enhance screen approach pathway?
there is barely enough A, but then reduciton of B creates or accentuates phenotype
What does the suppressor approach do when there is overactive sevenless and downstream mutation?
-it counteracts effects of the overactive sevenless
What happens in the enhancer approach at the restrictive temp vs the permissive temp?
-at the restrictive temp the sevenless gene doesnt form correctly so it is prone to knockout
-at the 22.7C the WT phenotype is very prone to downstream mutation
