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: