COCKBURN 2

Question 1

what is the characteristics of ced-1 mutants and the conclusion?

Answer 1

ced-1 mutants: cells die but they cannot be engulfed
dying corpses very easy to see
conclusion that ced-1 is required for engulfment

Question 2

what happens when you mutate both ced-1 and ced-3 and the conclusions that can be drawn from it?

Answer 2

mutated ced-3 in the background of ced-1; no cell death
ced-1 and ced-3 double mutants no longer have cell death
ced-3 is required for cell death
cell dead is an active process (there is a gene regulating it)
the worms are fine with the extra cells

Question 3

what happens when you mutate egl-1?

Answer 3

egl1 mutants: make eggs but cannot lay them
hermaphrodite specific neurons die (they are more sensitive to death signals and are required for laying eggs)
egl-1 worms make it easy to do screens=look for worms that can lay eggs again
identified another gene required for cell death: ced-4

Question 4

what are the characteristics of ced-9 mutants?

Answer 4

way too much cell death
ced-9 is required for cell survival
sequence is similar to Bcl2 gene in humans: B-cell lymphoma 2, which is an oncogene
Bcl2= more likely to get lymphoma

Question 5

how can Bcl-2 modulate cell death in c.elegans?

Answer 5

add a heat shock promoter before the ced-9/Bcl2 gene (activated when you heat shock it)
and you have a lot of extra cells, since they do not die

Question 6

what is the pathway of cell death of Bcl-2?

Answer 6

cell receives an intrinsic or extrinsic signal
1. normally, Bcl-2 sequesters and inhibits BAX and BAK
2. in death conditions, Bcl-2 releases them, BAX and BAK oligomerise and form a pore to permeabilise the mitochondrial membrane
3. mitochondrial cytochrome C is released , which activates caspase 9 (first in a cascade of proteins) via Apaf1
4. caspase 9 cleaves and activates caspase 3, which is the executioner and eats up everything, causing irreversible death

Question 7

how do the mutations fit into the Bcl-2 cell death pathway to alter it?

Answer 7

1. loss of Ced-9 (loss of Bcl-2) causes more cell death since BAX and BAK are always free
2. loss of ced-4(loss of Apaf1) causes less cell death because then you dont activate the cascade of proteases
3. loss of ced-3(loss of caspase 3) causes less cell death because you cannot chew up things
4. egl1 (worm specific) inhibits ced-9 and the original mutation is a gain of function (more egl-1 activity causes more cell death), ced-9 is inhibited so BAX and BAK are always free

Question 8

what are the different mutants in developmental timing?

Answer 8

wild type: goes through L1-4 till adulthood
lin14 (loss of function): skips L1 stage
lin14 (gain of function): only says in L1 stage
lin4: only stays in L1
lin4 identified as a repressor of lin14 (loss of lin4 leads to more lin14)

Question 9

what are the lin14 protein levels in different mutants?

Answer 9

wild type: a lot at first but then dies down
lin14 (lf): no protein
lin14 (gf): protein present always
lin4: protein present always

Question 10

how was it identified that lin4 does not code for a protein

Answer 10

try to look for open reading frames
but they were all too short
too small to code for a protein
compared with 4 species of worms: not conserved
clearly not important for protein coding

Question 11

how were Lin-4 transcripts investigated?

Answer 11

northern blotting (extract RNA and run on gel to separate by size)
blot on membrane
hybridise with radioactive nucleic acid probes of known sequences of lin-14
see what RNAs correspond to that and their size

Question 12

what are the two RNAs that Lin-4 encodes?

Answer 12

lin-4L (60bp) hairpin shape, folds back on itself
lin-4S (21bp) liner, complementary to lin14 UTR, and that sequence is also present in the long sequence
lin4 RNA is complementary lin14 mRNA= lin4 binds to lin14 and regulates lin14 levels

Question 13

what is another gene with a mechanism similar to Lin-4?

Answer 13

lin-7

Question 14

how were other miRNAs identified?

Answer 14

isolate 21bp RNAs from a gel
put them into plasmids to make a library and sequence them
all sit within 60bp similar hairpin forming sequences

Question 15

how are miRNAs processed?

Answer 15

in the nucleus drosha cuts the pri-microRNA into pre-microRNA (the 70bp hairpin)
through exportin 5 the pre-miRNA goes into the cytoplasm
dicer cleaves the pre-miRNA, cuts off the loop which creates the 21bp fragment
further processed by argonaute 2 and RISC

Question 16

what are the three different ways in which miRNA works?

Answer 16

binds to 3’UTR to downregulate the translation of that RNA;
1. can cause cleavage
2. can remove cap or poly A tail to make it more sensitive
3. can sit on it and prevent translation

Question 17

how is the complementarity of the miRNAs important?

Answer 17

not 100% complementary
complementary at the ends, but not necessarily in the middle
this means that one miRNA can bind to multiple targets and one mRNA target can be bound and regulated by multiple miRNAs (long UTR=lots of places)

Question 18

what type of regulatory mechanism are miRNAs in vertebrates?

Answer 18

“gentle”, used for fine tuning

Question 19

how are drosophila eyes organised?

Answer 19

organised in arrays of photoreceptors called ommatidia
always arranged in the same way
distal ommatidium: 7 receptors, R7 in the middle
proximal ommatidium: arrangement is the same but R7 becomes R8

Question 20

sev (sevenless) mutants

Answer 20

do not form R7
cannot recognise UV light
no clusters of R7 cells
the sev protein through immunofluorescence was localised to the plasma membrane: it could be a receptor

Question 21

boss mutants (bride of seven less)

Answer 21

also do not form R7
boss (all cells are mutant); no R7
boss (only R7 is mutant); a little bit of R7 (this means that R7 is still formed, R7 is required for the formation of R7 but it doesn’t have to be in R7)
boss (only R8 is mutant): no R7 (R8 is below R7, in contact with it)
generate mosaic flies (only a certain cell is mutant, not all)
reveals that Boss plays a cell non autonomous role in R7 development
=gene is important not in the cell that it is expressed in but in the cells nearby

Question 22

what is the relationship between Boss and sev?

Answer 22

sev is a receptor tyrosine kinase and boss is its ligand
boss is present in R8
sev is in R7: TM membrane receptor

Question 23

suppressor screens using constitutively active sev

Answer 23

sev (LOF): no R7
sev (GOF): extra cells, doesnt need ligand, constitutive
sev (GOF) and sos (LOF): sos returns things back to normal, which means it is downstream of sev

Question 24

what is the pathway of sev RTK?

Answer 24

boss binds to sev
in the R7 cell:
causes clustering of sev, autophosphorylation
binds to scaffold adaptor protein (drk)
binds to Ras-GEF (sos)
activates Ras by replacing GDP by GTP
active Ras now gives downstream signals

Question 25

what are transposons

Answer 25

mobile DNA elements

Question 26

what are the two classes of transposons?

Answer 26

class 1: copy paste
transcribe and then cDNA and then insert
class 2: cut and paste
code for what they need to excise themselves and can jump around

Question 27

how can you make the transposon work for you (P elements)

Answer 27

you can use PCR to identify where the P element landed (which gene is disrupted)
include a reporter gene (GFP or LacZ) to visualise where the targeted gene is normally expressed (gene trap)
if the P element contains fluorescent protein and promoter elements, insertion near enhancer will generate a reporter of enhancer activity (enhancer trap)
such as change in eye colour: unique to wild flies
put in plasmids and inject into flies
flies with a specific phenotype have the transposon