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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
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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: