Lecture 36

Question 1

describe how c elegans invariant embryogenesis can be further advanced

Answer 1

some ppl mapped every single lineage in the embryo & larva = can be combined with forward genetics to find genes important for development

Question 2

what do heterochronic mutants have

Answer 2

defects in developmental timing
larval cell divisions =
lin 14 loss of function = skips stage
lin 14 gain of function = frozen on first stages and same for lin 4
lin 4 protein levels = wt some, lin14 loss of function none, line 14 gain of function and lin 4 = many

Question 3

describe lin 4

Answer 3

does not code for protein
rna that seems to be regulating lin 4
look for orf
compared possible orfs across 4 species

Question 4

describe investigating lin 4 transcripts

Answer 4

northern blotting to detect lin 4 products = run on gel electrophoresis, probes: conserved lin 4 sequences, gives specific bands
lin 4 encodes 2 small rnas = when rescued lin4L 60bp hairpin, lin4S 21bp linear
lin 4 rnas are complementary to lin 14 mrna

Question 5

describe let 7

Answer 5

another gene with similar characteristics
Mutation is heterochronic
Northern blot detects a 21bp product
complementary to other heterochronic rnas
Could this be a common mechanism of gene regulation?

Question 6

what are micrornas

Answer 6

Everywhere including human cells
1- isolate 21bp rnas from a gel
2 - clone into plasmids
3 - sequence
Library of 22 mirnas from human (hela) cells
rnas all come from similar hairpin forming sequences
all sit in 60bp hairpion sequences = mechanism of regulation

Question 7

describe mirna processing and activity

Answer 7

transcribed
~70bp hairpin
21bp fragment ss
risc assembly = rna induced silencing complex
with argonaut 2
used to target rna that needs to be regulated
binds in 3’ utr of rna and downregulates

Question 8

what do mirnas recognize

Answer 8

complementary sequences
one mirna can bind multiple targets
one mrna target can be bound and regulated by multiple mirnas
vertebrates = mirnas tend to be a gentle regulatory mechanism important for fine tuning gene expression levels

Question 9

what do drosophila eyes contain

Answer 9

repeating arrays of photoreceptors - rods and cones
ommatidium
cross section of ommatidium
- distal = r7
- proximal = r8

Question 10

describe sevenless - sev mutants

Answer 10

do not form r7
missing cell
normal ommatidia tho
sevenless protein localizes to pm, early immunofluoresence staining = raise antibody that binds sevenless and secondary antibody that binds primary
no more clusters of sevenless observed

Question 11

describe bride of sevenless (boss)

Answer 11

mutants also do not form r7
boss - all cells mutant = make flies where boss missing only in some cells
boss - only r7 mutant -= boss needed nearby
boss -= only r8 mutant = r7 doesnt form
by generating mosaic flies boss can be deleted in specific subsets of cells
revealed that boss plays a cell non autonomous role in r7 development

Question 12

what is sev and what is boss

Answer 12

sev = rtkn and boss is its ligand
boss needs to be expressed in r8 to bind r7
sevenless = tm receptor for signalling pathway
supressor screens to find more muatnts

Question 13

describe supressor screens

Answer 13

using constitutively active sev
wild type
sev lof = loss of function - missing doesnt work properly, no r7 cells
sev gof = hyper activated, doesnt even need boss, extra r7 cells
sev gof sos lof (son of sevenless) = single r7 cell

Question 14

describe downstream components of sevenless rtk pathway

Answer 14

Activated sev rtk
adaptor proteins drk - sh2, sh3
recruits scaffold proteins = ras gef (sos) then
active ras protein bound gtp = downstream signals - important for r7 specification

Question 15

what are transposons

Answer 15

parasitic dna elements
jumping/mobile dna
ev and ltr transposons, non ltr transposons = class 1: copy and paste
dna transposons = class 2: cut and paste

Question 16

describe p element - gen

Answer 16

in wild flies, modify and insertion into fly and inject into fly germline then see which ones have transposons that move = look for transgenic flies with a specific phenotype

Question 17

what is advantage of p element

Answer 17

can use pcr to identify where p element landed - which gene is disrupted

Question 18

what is gene trap

Answer 18

can include reporter gene (fluorescent protein) to visualize where targeted gene is normally expressed
which cells express gene

Question 19

what is enhancer trap

Answer 19

if p element contains fluorescent protein and promoter elemnts
insertion near an enhancer will generate a reporter of enhancer activity
act with p element and express gfp

Question 20

describe lin 4 and lin 14

Answer 20

lin 14 mutants develop too quickly (loss of function) or too slowly (gain of function)
lin 4 identified as repressor of lin 14 (loss of lin 4 = more lin 14)