reading for monday’s lecture: ( genetic mosaics & chimeras )
DESCRIPTION
Reading for Monday’s lecture: ( genetic mosaics & chimeras ). p518 (“Aneuploid Mosaics…”) pp731-732 (“What cells…”). I will hold office hours during spring break, but at a different time than normal: during class time (11a-12N) on March 26 & March 30. - PowerPoint PPT PresentationTRANSCRIPT
Reading for Monday’s lecture: (genetic mosaics & chimeras)
p518 (“Aneuploid Mosaics…”)
pp731-732 (“What cells…”)
I will hold office hours during spring break, but at a differenttime than normal:
during class time (11a-12N) on March 26 & March 30
Using them to follow chromosomes in mutant screensBalancer chromosomes:
Aim: find genes that allow cells to know where they are so the cells can know what they should be
expected lof mutant phenotype for “pattern formation” genes:
(1) embryonic recessive lethal
(2) alterred dentical belt pattern (exoskeleton) in dead embryos
vvvvv
vv v
vv
vvvvv
vv
vv
vvvvv
vv v
vv
vvvvv
vv
vv
vvvvv
vv v
vv
vvvvv
vvv
vvvvvv
v
vvvvvv
v
vvvvv
vv
vvvvvvvv
v
wildtype
vvvvv
vv v
vv
vvvvv
vv
vvvvv
vvvv
vvvvvvv
vvvvvv
vvv
vv
vvvvv
vv
vv
vvvvv
vvv
vvvvvv
v
“bicaudal”
Post. Post.
(dying fly embryos can still differentiate a lot)
(genes generating positional information)
Ant. Post.polarity>>>
Consider the brute-force screen that led to the last fly Nobel Prize
N-V & W:
Second chromosome (brute force) screen
DTS / CyOfemales
cn bwmales
mutagenize
each son potentiallycarries a new mutant
allele of interest…but a different new mutant
in each
Second
take individual malesand mate separately
(10,000 crosses)
DTS / CyOfemales single
dominanttemperature-
sensitivelethal
second-chromosome
balancer second-chromosome“markers”(eye color = white)
CyO / cn bw & let??sons
X@non-permissive
DTS / CyOfemales single
CyO / cn bw & let??sons
X
CyO / cn bw let-a? CyO / cn bw let-a?sonsdaughters
Xeach group of progeny separately (forced incest):
CyO / CyO DTS / CyO
DTS/ cn bw let?
unwanted sibs all die
@non-permissive
CyO / cn bw let-a? cn bw let-a? / cn bw let-a? CyO / CyO
to maintain anynew let mutation
do they all die? (no white eyes?)and if so, when? how?
always die
only after a 2nd generation of 10,000 crossesdid they know which individual sons of mutagenized males
carried a recessive lethal mutation of interest (value)
CyO / cn bw let-a? CyO / cn bw let-a?sonsdaughters
Xeach group of progeny separately (forced incest):
Second chromosome screen
DTS / CyOfemales
cn bwmales
mutagenize
each son potentiallycarries a new mutant
allele of interest
CyO / cn bw mut-a? CyO / cn bw mut-a?
sonsdaughters
cn bw mut-a? / cn bw mut-a?
DTS / CyOfemales
single
X
CyO / cn bw & mut??
sons
X
X
only after a 2nd generation of 10,000 crossesdid they know which original F1 sons carried mutations of value
F1 generation
F2 generation
Brute force
keep populationsseparate!
…and if looking for maternal-effect mutations, go blindly one generation more!
Second chromosome screen
DTS / CyOfemales
cn bwmales
mutagenize
CyO / cn bw mut-a? CyO / cn bw mut-a?
sonsdaughters
CyO / cn bw mut-a? cn bw mut-a? / cn bw mut-a? CyO / CyO
DTS / CyOfemales
single
X
sons
X
XCyO / cn bw & mut??
@non-permissive
@non-permissiveOR permissive
DTS / cn bw & mut??
or
Temperature forfirst cross doesn’t
really matter:
(1) have to hand-pick males anyway
(2) males have nomeiotic recombination (so DTS/mut OK)
Classic N-V&W screen illustrates two important points:
(1) recessiveness (~lof) generally demands multiple generations of blind forced incest crosses (mating siblings) to recover mutant
(2) recognizing an informative phenotype is a large part of the genetics game
The N-V & W advantage: an informative phenotype that could be scored in dead embryos (didn’t demand survival -- or much else!).
What if want to study something like eye development instead?
ey1 :recessive hypomorph, adults w/ no eyes
ey-(null) : recessive embryonic lethaley is pleiotropic
(multiple “unrelated”phenotypes/functions)
…can we overcome the limitations of recessiveness?
Attractive features: interesting AND non-essential (and more), but consider:
…can we overcome the limitations of pleiotropy?
Got lucky with ey1
how many other important eye genes missed?
&Early
YES…we shall overcome
(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)
(2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!)
but first: already mentioned one way to deal with pleiotropy
temperature-conditionalmutant alleles
ts muts. waytoo limited
evenin
flies& worms
FARBETTER
…can we overcome the limitations of pleiotropy?
(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)
sevenless/+ (wildtype)vs. sev/sev R7 photoreceptor
missing(turned into cone cell)
Illustrate with example from fly eye development studies:
?conecell
photo-recptrphoto-recptr
conecell
signal fromR8 neighbor
A developmental decision:
R7 precursor cell
Stemmed from original observation:
goal: make genes “artificially” haploinsufficient
bride-of-sevenless (null eye-specific)
seven-in-absentiason-of-sevenless
seven-up
Other genes discoveredto be involved in theR7 precursor decision:
null sev allele: same thing(hence, eye-specific)
null alleles not eye-specific:pleiotropic:
How many other pleiotropic genes missed?
(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)
sev/sevR7 photoreceptor
missing(turned into cone cell)
?conecell
photo-recptrphoto-recptr
conecell
signal fromR8 neighbor
R7 precursor cell
make genes “artificially” haploinsufficient
How many pleiotropic genes missed?sev- /sev- ; TM3,P{sevB4(ts)}/+
designer ts allele
sev encodes v-src homolog (human oncogene)
screen for dominant mutations that make:
24.3oC
22.7oC
R7 absent
R7 present
R7 present (Dominant suppressors)
R7 absent (Dominant enhancers)
growthtemperature
phenotype
(3rd chromosome balancer)
(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)
screen for dominant mutations that make:
24.3oC
22.7oC
R7 absent
R7 present
R7 present (Dominant suppressors)
R7 absent (Dominant enhancers)
growthtemperature
phenotype
Found many pleiotropic lof alleles of both types (incl. recessive lethals).
Poising sev+ activity level on a phenotypic threshold made other genes haploinsufficient
Wildtype fly must normally have an excess of sev+ activity as insurance, so it can tolerate fluctuations in levels of other genes in pathway during development
…if take away that cushion, now more sensitive to reductions in other gene levels
but only with respect to sev function!
sevenless/+ (wildtype)vs. sev/sev R7 photoreceptor
missing(turned into cone cell)
made genes “artificially” haploinsufficient
R7 precursor cell
photo-recptr
conecell
signal fromR8 neighbor
….then look for newly induceddominant enhancer or suppressor alleles
adjust level to poise systemon phenotypic threshold
sevenless/”+“
other genes inpathway NOThaploinsufficient
now other genes in pathway
ARE haploinsufficient
Point to keep in mind:…will not necessarily identify every relevant gene in pathway this way
sevenless: receptor in R7 cell that responds to signal from R8
bride-of-sevenless: ligand (signal molecule) generated in R8
no new mutant alleles foundin sev sensitized screen!
(2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!)
…recover new recessives in the F1???
Based on a phenominon discovered (‘30s) by former chairof U.C. Zoology Dept: mitotic recombination
…only possible because of a very strange aspect offly chromosome behavior:
homologous chromosomes pair during mitotic interphase
but improved upon enormously in modern times
Another way around the limitations of pleiotropy in genetic screens: