Steps in forward genetics:

generate informative mutant alleles

recover informative mutant alleles

study informative mutant allele (do molecular biology)

write paper

reap rewards

decide what to study

NATURE V287:p795 (1980) Nobel Prize 1995

wildtype

wildtype

gooseberry

gooseberry

patch

patch

mutant phenotypes

informative for understanding

pattern formation in metazoans

“skins” ofdead larvae

text: 20.3 “The genetic analysis of body-plan development

in Drosophila: a comprehensive example. (pp732-745)

Fig. 20.26 p744Fig. 20.22 p741

the segmentation regulatory gene hierarchy

Ant. Pst.

gap

pair-

rule

segment

polarity

maternal

effect

Mammalian Hox gene clusters

Mouse embryo

Drosophila homeotic gene clusters

They regulate…

Two general categories of mutant allele recovery strategies:

(1) genetic screens

make mutations randomly, then

you sift through chromosomes (often one at a time)

looking for mutant alleles of interest/use

(2) genetic selections

make mutations randomly, then

let nature eliminate all undesired mutant alleles

so you are only left with the good stuff

but often not possible & potentially more biased2 easier than 1,

(“brute force” screens)

generate mutant allele

By the way:

an important but under-appreciated step in genetic analysis:

recover mutant allele

study mutant allele

write paper

reap rewards

maintain mutant allele

generate mutant allele

recover mutant allele

study mutant allele

write paper

reap rewards

Homework problem:

How much food (corn meal, molasses, yeast) has

T.H.Morgan’s original white1 mutant line consumed

since 1910?

for each fly line: transfer to 15ml new food every 3 weeks

Strategies & tools that help us recover mutant alleles

can also help us maintain them.

Maintaining mutant stocks (lines) in model genetic systems:

most microbes(spores are nice)

arabidopsis

(“the weed”)

& corn

seeds

mouse

fish

fly

worm

(embryos)

To freeze or not to freeze

Basic facts to consider

in designing screens and selections:

(1) Most LOF mutant alleles are recessive (all else being equal)

(LOF mutations are the most frequent class)

(2) Most null alleles of genes with an obvious LOF phenotype

are lethal, or at least sterile.

(3) Most “developmentally interesting” genes are

essential for viability or fertility

Hence:

screen/selection schemes must provide for the recovery of

recessive lethals and steriles

The “diploid advantage”

for recessive lethal studies:

(+ holds the fort)

(let naked and exposed)

Diploid:

lethal / + alive (fertile)

Haploid:

lethal dead (sterile)

rely on conditional lethals in generating mutations:

oftenfor microbes

all grow

(mutagenize

wildtype)

only mutants of interest don’t grow

growth vs. no growth

condition A condition B

Two key tricks for microbes:

(let naked and exposed)

Haploid:

lethal dead (sterile)

rely on conditional lethals in generating mutations:

p212: Fig. 7.5 Replica Plating

& p558: Fig. 15.15

p547: Fig. 15.5 (Penicillin) enrichment

augmented by:genetic

selection

mutantscreen orselection?

geneticscreen

all grow

(mutagenized)only mutants of

interest don’t grow

growth vs. no growth

condition A condition B

Two key tricks for microbes:

p212: Fig. 7.5

& p558: Fig. 15.15

geneticscreen

Replica Plating

all grow

(mutagenized)only mutants of

interest don’t grow

growth vs. no growth

condition A condition BTwo key tricks for microbes:

p212: Fig. 7.5

& p558: Fig. 15.15

Replica Plating

geneticselection

p547: Fig. 15.5 (Penicillin) enrichmentaugmented by:

geneticscreen

Replica plate on

(diluting outthe penicillin)

The “diploid advantage”

for recessive lethal studies:

(+ holds the fort)

(let naked and exposed)

Diploid:

lethal / + alive (fertile)

Haploid:

lethal dead (sterile)

The “diploid handicap”

for recessive lethal studies:

+ masks lethal

effects oflethal

immediatelyobvious

The problem with diploids in hunting for new recessive mutations:

+

++

++

+

+

eggs

+a

b

c

d+

++

+ +

++

sperm

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

:PARENTS

+/+

+/+a+/

b+/

PROGENYF1

a

b

++

+

+

++

form zygotes

The problem with diploids in hunting for new recessive mutations:

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/ F1 PROGENY

…the individual who can produce a-/a- offspring.

How do we know who (if anyone) is carrying a- ?

given:we are interested in(finding) the a-/a- phenotype

The problem with diploids in hunting for new recessive mutations:

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/ F1 PROGENY

How do we know who (if anyone) is carrying a- ?

given:we are interested inthe a-/a- phenotype

…the individual who can produce a-/a- offspring.

To whom do we mate to find out?

If we can “self” this individual,

we are effectively mating to +/a- for sure

YES! & we know in the F2of course, we hadto self everyone: No a-/a-

The problem with diploids in hunting for new recessive mutations:

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/To whom do we mate

to find out -- if we can’t self?

+/+

+/+

+/+a+/

+/+a+/

…. we still don’tknow in the F2!

+/+

Meanwhile+/+

+/+

+/+

+/+

+/+

F1

The problem with diploids in hunting for new recessive mutations:

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/

-- if we can’t self?

+/+

+/+a+/

+/+a+/

…. we still don’tknow in the F2!

+/+

+/+

+/+

+/+

+/+

F1

..but at leastnow we havepotential mates with a-

F2

a /aat least some chance:+/+at best

Mateinter seMate

inter se

X+/+ X+/+(must keep populations

separate!)

The problem with diploids in hunting for new recessive mutations:

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/

-- if we can’t self?

+/+

+/+

a+/

+/+

a+/

+/+

+/+

+/+

+/+

+/+

F1

F2

a /aif we cross them,

a-/a- will come:+/+only

can wedo better

thanmating

inter se?Mateinter se

+/+X

mutagenize(altv.: dysgenic)

Female MaleX+/+ +/+

+/+ +/+ a+/b+/

+/+

+/+

a+/

+/+

a+/

F1

F2

a /aif we cross them,

a-/a- will come:

we cando better

thanmating inter se

+/+X

+ = mutagenized but not desired mutant

+ = non-mutagenized from original Mom

+ = non-mutagenized from F1 mate

a- = mutagenized chromosome with new mut.

It would help if we could keep track of chromosomes:

Even nicer

if we could

eliminate the

extraneous animals

our friend, Herman Muller had the answer (early ‘30s):

Balancer chromosomes:

(a) a chromosome you can distinguish from the others.

(b) a chromosome that will not recombine with others

(c) a chromosome that will not “become” homozygous (i.e. that would either be lethal or sterile if homozygous)

dominant marker mutant alleles (Bar, Curly, Stubble)

crossover suppressors (multiple inversions)

recessive lethal or sterile alleles

(1)used them to determine mutation frequency:

…how often a new recessive lethal arose on a given fly chromosome

(2) used them to “maintain” deleterious recessive alleles of interest

p503: Fig. 14.17

Balancer chromosomes:

(a) dominant marker mutant alleles

(b) crossover suppressors

(c) recessive lethal or sterile alleles

(1) use them to follow chromosomes in order to create

new genotypes (such as in mutant screens)

(2) use them to “maintain” deleterious recessive alleles of interest

3 essential features:

Balancer chromosomes:

(2) used them to “maintain” deleterious recessive alleles of interest

with balancer: let/Bal

let +-A- -A DomDom+ let - Inv-Blet-B+let /Bal,

let-A-/Bal let-A-/BalX let-A-/let-A- lethal

let-A-/Bal O.K.

Bal/Bal lethal

Balanced lethal condition

progenyjust like parentsby default

let/+ let/+ +/+ let/+ let/letX & &

+/+ let/+&

recall problem without balancer:

+/+Xlet/++/++/+ +/+X

Balancer chromosomes:

(2) used them to “maintain” deleterious recessive alleles of interest

What about an X-linked recessive lethal?

lethal

let-A-/Bal O.K. female

Bal/Bal sterile female

Balanced condition

lethalor sterile

recessive

female-specific

sterile

/Y

let-A-/Y lethal male

Bal /Y O.K. male

let-A-/Bal let-A-/YX or Bal /Y

female male

(1) use them to follow chromosomes in mutant screens

Balancer chromosomes:

Aim: find genes that allow cells to know where they are

so that those 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

vvv

vvv

v vvv

vvv

v vv

vvv

vvv

v vv

v vvv

vvv

v vv

vvv

vvv

vvv

vv

vv

vvv

v vv v

v

vvvv

vvv

vvv v

v vv

vvv

v vv

vvv

vvvv

vvv

wildtype

vvv

vvv

v vvv

vvv

vvv

vv v

vvv

vvv v

v

vvv v

v vvvv

vv v

vvv

v v

vvv

v vv

vvv

vvv

vv

vvv

vvv v

v vv

“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: