1. your favorite gene(s) & w+
How to get fly transgenes from in vitro to in vivo:
your favorite gene(s) & w+
transposase gene
A “stable” source of
transposase,
since it can’t move
P-element (ends) serve as
a “vector” to move DNA
of our choice
As a GENETIC SOURCE of transposase
to get the transgene to insert into chromosomes
use a defective (immobile) integrated P-element
Inject DNA into very young embryos
….. aiming for the germ cells.
modified so that it makes transposase
in soma as well as germline

2. whatever is useful (eg. w+)
Use the engineered mobile genetic element
as a mutagen (make it hop randomly into genes):
whatever is useful (eg. w+)
(a nonautonomous element)
starting element: w+ 2
It is a "genetically tagged" mutagen (we can follow it)

3. transposase to mobilize
(pseudo-replicative
transposition) X Tp
new (random) site of insertion w+ w+
starting element: 2
happening
In the dysgenic
parent's germline 3
collect progeny from the
appropriate genetic cross
of the dysgenic parent X
(not carrying the transposase source) 2
(not carrying the original P) 3
gene w+
If so, already well marked
and easy to clone!
Is this a mutant allele of interest?

4. your favorite gene(s) & w+P P
your favorite gene(s) & w+
(a nonautonomous element)
If we are going to want to use as a mutagen (hop into genes)…
Lucky thing that M strains exist
(strains with no pre-existing source of P transposase or antitransposase
to interfere with our controlling non-autonomous element [transgene] mobility)

5. Lab strains taken from the wild before 1950’s don’t have P elements.
Why do M strains exist?
Lab strains taken from the wild before 1950’s
don’t have P elements.
This DNA parasite invaded D. melanogaster
sometime in the 1950s, then rapidly spread
nearest P-element relative: in a fly species 50 Myr diverged
-- an example of horizontal genetic transmission
How?
(xfrd in same generation, i.e. gene not introduced from parents (vs. vertical)
Are fly-workers to blame for contaminating D. melanogaster?

6. recover the chromosome in a MUTANT SCREEN or SELECTION
tagged transposon hopped in the
dysgenic parent's
germline
collect progeny from the
dysgenic parent w+ 3
gene
new (random) site of insertion
How do we know that we have even mutated a gene,
much less generated a mutant allele of use/interest ?
recover the chromosome in a
MUTANT SCREEN or SELECTION

7. Steps in forward genetics:
decide what to study
generate informative mutant alleles
(mutagenesis)
recover informative mutant alleles
study informative mutant allele (do molecular biology)
write paper
reap rewards

8. NATURE V287:p795 (1980)
Nobel Prize 1995
(products of a
mutant screen)

9. informative for understanding pattern formation in metazoans
wildtype
gooseberry
wildtype
gooseberry
patch
“skins” of
dead larvae
patch
mutant phenotypes
informative for understanding
pattern formation in metazoans

10. text: 20.3 “The genetic analysis of body-plan development
in Drosophila: a comprehensive example. (pp )

11. They regulate… Fig. 20.22 p741 Fig. 20.26 p744
Mammalian Hox gene clusters
Mouse embryo
Drosophila homeotic gene clusters
the segmentation regulatory gene hierarchy
Ant.
Pst.
gap
pair-
rule
segment
polarity
maternal
effect
Fig p741
Fig p744

12. Two general categories of mutant allele recovery strategies:
(“brute force” screens)
(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
2 easier to execute than 1,
but often not possible to design
& potentially more biased
(may get only what you think to look for)

13. an important but under-appreciated step in genetic analysis:
By the way:
an important but under-appreciated step in genetic analysis:
maintain mutant allele
generate mutant allele
recover mutant allele
study mutant allele
write paper
reap rewards
generate mutant allele
recover mutant allele
study mutant allele
write paper
reap rewards
for each fly line:
transfer to 15ml new food
every 3 weeks
Homework problem:
How much food (corn meal, molasses, yeast) has
T.H.Morgan’s original white1 mutant line consumed
since 1910?
Strategies & tools that help us recover mutant alleles
can also help us maintain them.

14. Maintaining mutant stocks (lines) in model genetic systems:
To freeze or not to freeze
most microbes
mouse
(embryos)
(spores are nice)
fish
arabidopsis
(“the weed”)
& corn
fly
seeds
worm

15. 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

16. The “diploid advantage” for recessive lethal studies:
alive (fertile)
(+ holds the fort)
Haploid:
lethal
dead (sterile)
rely on conditional lethals in generating mutations:
often
for microbes
(let naked and exposed)

17. genetic screen Haploid: lethal dead (sterile)
rely on conditional lethals in generating mutations:
growth vs no growth
condition A
condition B
genetic
screen
mutant
screen or
selection?
all grow
(mutagenize
wildtype)
only mutants of
interest don’t grow
Two key tricks for microbes:
p212: Fig Replica Plating
& p558: Fig
p547: Fig (Penicillin) enrichment
augmented by:
genetic
selection

18. Replica Plating genetic screen growth vs. no growth condition A
condition B
all grow
(mutagenized)
only mutants of
interest don’t grow
Two key tricks for microbes:
p212: Fig. 7.5
& its use: Fig (p558)
Replica Plating
genetic
screen

19. augmented by: p547: Fig. 15.5 (Penicillin) enrichment
growth vs no growth
condition A
condition B
all grow
(mutagenized)
only mutants of
interest don’t grow
genetic
selection
p547: Fig (Penicillin) enrichment
augmented by:
Replica plate on
(diluting out
the penicillin)

20. The “diploid handicap” for recessive lethal studies:
The “diploid advantage”
for recessive lethal studies:
(+ holds the fort)
(let naked and exposed)
Diploid:
lethal / +
alive (fertile)
Haploid:
lethal
dead (sterile) +
masks
lethal
effects of
immediately
obvious

21. The problem with diploids in hunting for new recessive mutations:+
form zygotes
mutagenize
Female
Male X
+/+
:PARENTS +
eggs + a b c d
sperm
+/+ a +/ b
PROGENY F1

22. The problem with diploids in hunting for new recessive mutations:
mutagenize
Female
Male X
+/+
+/+ b +/
+/+ a +/
F1 PROGENY
given:
we are interested in
(finding) the a-/a- phenotype
How do we know who (if anyone) is carrying a- ?
…the individual who can produce a-/a- offspring.

23. The problem with diploids in hunting for new recessive mutations:
mutagenize
Female
Male X
+/+
Which is the individual
who can produce a-/a- offspring?
of course, we had
to self everyone: No a-/a-
+/+ b +/
+/+ a +/
F1 PROGENY
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 F2
a-/a-

24. The problem with diploids in hunting for new recessive mutations:
mutagenize
Female
Male X
+/+ F1
+/+ b +/
+/+ a +/
To whom do we mate
to find out -- if we can’t self?
+/+
Meanwhile
+/+
…. we still don’t
know in the F2!
+/+
+/+ a +/

25. a /a The problem with diploids in hunting for new recessive mutations:
-- if we can’t self.
mutagenize
Female
Male X
+/+
…. we still don’t
know in the F2! F1
+/+ b +/
+/+ a +/
..but at least now we have
potential mates with a-
male?
female?
X+/+
X+/+
(must keep
populations
separate!)
+/+ F2
+/+ a +/
Mate
inter se
+/+
at best
a /a
at least some chance:

26. a /a The problem with diploids in hunting for new recessive mutations:
-- if we can’t self.
mutagenize
Female
Male X
+/+ F1
+/+ b +/
+/+ a +/
+/+ X a +/ F2 a +/
+/+
+/+
can we
do better
than
mating
inter se?
+/+
a /a
if we cross them,
a-/a- will come:
+/+
Mate
inter se
+/+
only

27. a /a + = mutagenized but not desired mutant
It would help if we could keep track of chromosomes:
a- = mutagenized chromosome with new mut.
+ = non-mutagenized from original Mom
Male
mutagenize X
+/+
+ = mutagenized but not desired mutant
Female
+/+
+ = non-mutagenized from F1 mate
+/+ a +/ b F1
+/+ X
a /a
if we cross them,
a-/a- will come:
+/+ a +/ F2
Even nicer
if we could
eliminate
extraneous animals
we can
do better
than
mating
inter se

28. Balancer chromosomes:
our friend, Herman Muller had the answer (early ‘30s):
(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
Balancer chromosomes:
(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 will not “become” homozygous
(i.e. that would either be lethal or sterile if homozygous)
recessive lethal or sterile alleles