Download CyO / cn bw let-a?

Reading
on inversions and their genetic consequences
pp501-503
genetic mosaics:
pp152-4 (Mitotic recombination…and cancer)
p518 (“Aneuploid Mosaics…”)
pp731-2 (“What cells…”)
I'll be holding office hours (once) during spring break
and at a special time: Wed, March 26, 11a-12N
I’ve asked to reserve a room from 7-9pm Wednesday,
April 9, for a review session in connection with
the midterm on Monday, April 14. I’ll announce where the
session will be held as soon as I know myself.
In connection with genetic screens & selections:
it would help if we could keep track of chromosomes
and eliminate extraneous animals so that we don't have
to rely on random inter se matings for the F2 cross:
mutagenize
Female X Male
+/+
+/+
+/+
+/b
+/+
F1
+/ a
X +/+
+/ a
F2
+/+
+/ a
+/+
+/+
a /a
Herman Muller gave fly workers Balancer chromosomes
for this purpose (early ‘30s - '40s):
…not only to facilitate mutant screens (and selections)
but also to
faciliate the maintenance of the deleterious alleles recovered
in such screens and selections.
Three key features:
(a) a chromosome you can distinguish from the others.
dominant marker mutant alleles (Bar, Curly, Stubble)
(b) a chromosome that will not recombine with others
crossover suppressors (multiple inversions)
(c) a chromosome that cannot become homozygous
recessive lethal or sterile alleles
p503: Fig. 14.17
(read about inversions
and their genetic consequences
on pp501-503)
Balancer chromosomes:
(2) use them to “maintain” deleterious recessive alleles of interest
…hence parents
must have carried let
and some siblings do
Problem without balancer:
let/+ X let/+
+/+ & let/+ & let/let
Must rely on matings with the let carriers
to maintain the let allele.
Balancer chromosomes:
(2) use them to “maintain” deleterious recessive alleles of interest
Problem without balancer:
let/+ X let/+
+/+ & let/+ & let/let
let/+ X let/+
+/+ & let/+ & let/let
Three
possible
let/+ X
random
sib matings:
+/+
+/+ X +/+
+/+ & let/+
+/+
…but with a balancer chromosome:
let/Bal =
let -A- let-B+ Dom+ /Bal, let-A+ Dom let -B- Inv
let-A-/Bal X let-A-/Bal
progeny
just like parents
by default
let-A-/let-A- lethal
let-A-/Bal
O.K.
Bal/Bal
Balanced lethal condition
lethal
Balancer chromosomes:
(2) used them to “maintain” deleterious recessive alleles of interest
What about an X-linked recessive lethal?
female
let-A-/Bal
male
recessive
female-specific
sterile
X let-A-/Y or Bal /Y
lethal
Balanced condition
lethal
or sterile
let-A-/Bal
O.K. female
Bal/Bal
sterile female
let-A-/Y
lethal male
Bal /Y
O.K. male
Balancer chromosomes:
(1) use them to follow chromosomes in mutant screens
Consider the brute-force screen that led to the last fly Nobel Prize
N-V & W:
Aim: find genes that allow cells to know where they are
so the cells can know how they should differentiate
expected lof mutant phenotype for “pattern formation” genes:
(genes generating
positional information)
(1) embryonic recessive lethal
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
vvvvvvvvvv
wildtype
polarity>>>
vvvvvvvvv
Post.
vvvvvvvv
Ant.
vvvvvvv
vvvvvvv
vvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
(2) alterred dentical belt pattern (exoskeleton)
in dead embryos (dying fly embryos can still differentiate a lot)
Post.
Post.
“bicaudal”
Balancer chromosomes:
Using them to follow chromosomes in mutant screens
Consider the brute-force screen that led to the last fly Nobel Prize
N-V & W:
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:
(genes generating
positional information)
(1) embryonic recessive lethal
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
vvvvvvvvvv
wildtype
polarity>>>
vvvvvvvvv
Post.
vvvvvvvv
Ant.
vvvvvvv
vvvvvvv
vvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
(2) alterred dentical belt pattern (exoskeleton)
in dead embryos (dying fly embryos can still differentiate a lot)
Post.
Post.
“bicaudal”
Second
Second chromosome (brute force) screen
dominant
secondtemperature- chromosome
sensitive
balancer
lethal
DTS / CyO
mutagenize
X
females
cn bw
males
@non-permissive
temp. for progeny
DTS / CyO
females
X
CyO / cn bw
single sons
second-chromosome
“markers”
(eye color = white)
each son potentially
carries a new recessive mutant
allele of interest
…but a different new mutant
in each
& let??
take individual males
and mate separately
(10,000 crosses)
DTS / CyO
females
@non-permissive
CyO / cn bw let-a?
daughters
X
CyO / cn bw & let??
single sons
X
CyO / cn bw let-a?
sons
each group of progeny from a particular
male are kept separate
(forces incest)
unwanted sibs all die
CyO / CyO
DTS / CyO
DTS/ cn bw let?
CyO / cn bw let-a?
daughters
cn bw let-a?
X CyO /sons
The progeny from forced incest:
CyO / cn bw let-a?
cn bw let-a? / cn bw let-a?
to maintain any
do they all die? (no white eyes?)
new let mutation in a
and if so, when? how?
balanced lethal condition
CyO / CyO
always die
only after a 2nd generation of 10,000 crosses
did they know which individual sons of mutagenized males
carried a recessive lethal mutation of interest (value)
Second chromosome screen
mutagenize
DTS / CyO
females
Brute force
cn bw
X
males
each son potentially
carries a new mutant
allele of interest
DTS / CyO
females
X
CyO / cn bw & mut??
F1 generation
single sons
CyO / cn bw mut-a?
daughters
cn bw mut-a?
X CyO / sons
cn bw mut-a? / cn bw mut-a?
F2 generation
keep populations
separate!
only after a 2nd generation of 10,000 crosses
did they know which original F1 sons carried mutations of value
…and if looking for maternal-effect mutations, go blindly one generation more!
Temperature for
first cross doesn’t
really matter:
mutagenize
DTS / CyO
females
X
@non-permissive
OR permissive
DTS / CyO
females
X
cn bw
males
(1) have to handpick males anyway
CyO / cn bw & mut??
single
sons
or
(2) males have no
meiotic recombination
(so DTS/mut OK)
DTS / cn bw & mut??
@non-permissive
Either way, this is all we get.
CyO / cn bw mut-a?
daughters
cn bw mut-a?
X CyO / sons
CyO / cn bw mut-a?
cn bw mut-a? / cn bw mut-a?
CyO / CyO
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
…can we overcome the limitations of recessiveness?
(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!). &Early
What if want to study something like eye development instead?
What if want to study something like eye development instead?
Attractive features: interesting AND non-essential (and more), but consider:
ey1 :recessive hypomorph, adults w/ no eyes
ey-(null) : recessive embryonic lethal
Got lucky with ey1
ey is pleiotropic
(multiple “unrelated”
phenotypes/functions)
how many other important eye genes missed?
…can we overcome the limitations of pleiotropy?
…can we overcome the limitations of pleiotropy?
YES…we shall overcome
but first:
already mentioned one way to deal with pleiotropy
temperature-conditional
mutant alleles
FAR
BETTER
ts muts. way
too limited
even
in
flies
& worms
(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!)
(1) genetically sensitize the system:
turn lof recessives into dominants (but only with respect to
one non-essential aspect of the genes’ function)
goal: make genes “artificially” haploinsufficient
Illustrate with example from fly eye development studies:
One of many cell fate decisions
made during eye development:
R7 precursor cell
signal (from
?
photo- R8 neighbor)
cone photorecptr
cell recptr
R7 precursor cell
signal from
?
photo- R8 neighbor
cone photorecptr
cell recptr
Other genes discovered
to be involved in the
R7 precursor decision:
The observation that started it all:
sevenless/+ (wildtype)
vs. sev/sev R7 photoreceptor
missing
(turned into cone cell)
sev mutant allele was a null
(hence, eye-specific)
bride-of-sevenless (null eye-specific)
null alleles not eye-specific:
pleiotropic: son-of-sevenless
seven-in-absentia
seven-up
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)
make genes “artificially” haploinsufficient
Isolate mutant alleles that interfere with eye development
but do not disrupt other (perhaps essential) functions
that some genes may have.
R7 photoreceptor
missing
sev/sev
(turned into cone cell)
sev encodes v-src homolog
(human oncogene)
(3rd chromosome balancer)
sev- /sev- ; TM3, P{sevB4(ts)} /+
designer ts allele
modeled after ts human allele
growth
temperature
phenotype
screen for dominant mutations
that make:
24.3oC
R7 absent
R7 present (Dominant suppressors)
22.7oC
R7 present
R7 absent (Dominant enhancers)
Clearly at either of these two temperatures, the system
governing the R7 decision is poised on a phenotypic threshold
growth
temperature
phenotype
screen for dominant mutations
that make:
24.3oC
R7 absent
R7 present (Dominant suppressors)
22.7oC
R7 present
R7 absent (Dominant enhancers)
Found many pleiotropic lof alleles of both types IN AN F1 GENETIC SCREEN:
dominant enhancers or suppressors of the R7 phenotype.
But many of these DOMINANT "modifiers" were also recessive lethal
(pleiotropic -- had other essential functions).
Poising sev+ activity level on a phenotypic threshold made other
genes haploinsufficient but only with respect to sev function!
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
R7 precursor cell
signal from
cone photo- R8 neighbor
cell recptr
Asked:
what genes work with sevenless to control this developmental decision?
Found those other genes by making them “artificially” haploinsufficient
with respect to the sev function, but
NOT haploinsufficient with respect to other functions that they might have.
now other genes
in pathway
other genes in
ARE
pathway
NOT
haploinsufficient
haploinsufficient
sevenless/”+“
adjust level to poise system
on phenotypic threshold
….then can look for newly induced
dominant enhancer or suppressor alleles
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 found
in sev sensitized screen!