Transcript /+ +/+ +/+ +/+ a +/ b - Molecular and Cell Biology

How to get fly transgenes from in vitro to in vivo:
your favorite gene(s) & w+
P-element (ends) serve as
a “vector” to move DNA
of our choice
Inject DNA into very young embryos
….. aiming for the germ cells.
As a GENETIC SOURCE of transposase
to get the transgene to insert into chromosomes
use a defective (immobile) integrated P-element
transposase gene
A “stable” source of
transposase,
since it can’t move
modified so that it makes transposase
in soma as well as germline
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)
transposase to mobilize
(pseudo-replicative
transposition)
Tp
X
starting element:
w+
2
w+
new (random) site of insertion
happening
In the dysgenic
parent's germline
3
collect progeny from the
appropriate genetic cross
of the dysgenic parent
(not carrying the transposase source)
X
(not carrying the original P)
2
w+
gene
3
Is this a mutant allele of interest?
If so, already well marked
and easy to clone!
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)
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
How? -- an example of horizontal genetic transmission
(xfrd in same generation, i.e. gene not introduced from parents (vs. vertical)
Are fly-workers to blame for contaminating D. melanogaster?
tagged transposon hopped in the
dysgenic parent's
germline
collect progeny from the
dysgenic parent
w+
gene
new (random) site of insertion
3
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
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
NATURE V287:p795 (1980)
Nobel Prize 1995
(products of a
mutant screen)
wildtype
gooseberry
wildtype
gooseberry
patch
“skins” of
dead larvae
patch
mutant phenotypes
informative for understanding
pattern formation in metazoans
text: 20.3 “The genetic analysis of body-plan development
in Drosophila: a comprehensive example. (pp732-745)
They regulate…
the segmentation regulatory gene hierarchy
Ant.
Pst.
maternal
effect
Drosophila homeotic gene clusters
Mammalian Hox gene clusters
gap
Mouse embryo
pairrule
segment
polarity
Fig. 20.22 p741
Fig. 20.26 p744
Two general categories of mutant allele recovery strategies:
(1) genetic screens
(“brute force” 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)
By the way:
an important but under-appreciated step in genetic analysis:
generate mutant allele
recover mutant allele
study mutant allele
maintain mutant allele
write paper
for each fly line:
transfer to 15ml new food
every 3 weeks
reap rewards
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.
Maintaining mutant stocks (lines) in model genetic systems:
To freeze or not to freeze
most microbes
(spores are nice)
arabidopsis
(“the weed”) seeds
& corn
worm
mouse
fish
fly
(embryos)
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:
Diploid:
lethal / +
alive (fertile)
(+ holds the fort)
Haploid:
often
for microbes
lethal
dead (sterile)
(let naked and exposed)
rely on conditional lethals in generating mutations:
Haploid: lethal
dead (sterile)
rely on conditional lethals in generating mutations:
condition A
growth
all grow
(mutagenize
wildtype)
vs.
condition B
no growth
only mutants of
interest don’t grow
mutant
genetic
screen or
screen
selection?
Two key tricks for microbes:
p212: Fig. 7.5
Replica Plating
& p558: Fig. 15.15
augmented by:
genetic
p547: Fig. 15.5 (Penicillin) enrichment selection
condition A
growth
vs.
all grow
(mutagenized)
condition B
no growth
only mutants of
interest don’t grow
Two key tricks for microbes:
p212: Fig. 7.5
& its use: Fig. 15.15 (p558)
Replica Plating
genetic
screen
condition A
growth
all grow
(mutagenized)
vs.
condition B
no growth
only mutants of
interest don’t grow
augmented by:
p547: Fig. 15.5 (Penicillin) enrichment
genetic
selection
(diluting out
the penicillin)
Replica plate on
The “diploid advantage”
for recessive lethal studies:
Diploid:
lethal / +
alive (fertile)
(+ holds the fort)
The “diploid handicap”
for recessive lethal studies:
+
masks
lethal
Haploid:
lethal
dead (sterile)
(let naked and exposed)
effects of
lethal
immediately
obvious
The problem with diploids in hunting for new recessive mutations:
mutagenize
Female X Male
+/+
+/+
:PARENTS
form zygotes
+
b
+
+
a
+
+
+
+
+
eggs
+
+ +
d
+
+
+
b
+
+ +
c
a
+ +
+
+/+
sperm
+
+/b
+/+
+/ a
F1 PROGENY
The problem with diploids in hunting for new recessive mutations:
mutagenize
Female X Male
+/+
+/+
+/+
+/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.
The problem with diploids in hunting for new recessive mutations:
mutagenize
Female X Male
+/+
+/+
+/+
+/b
+/+
Which is the individual
who can produce a-/a- offspring?
+/ 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
of course, we had
to self everyone: No a-/a-
YES! & we know in the F2
a-/a-
The problem with diploids in hunting for new recessive mutations:
mutagenize
Female X Male
+/+
+/+
F1
+/+
+/b
+/+
+/ a
To whom do we mate
to find out -- if we can’t self?
+/+
+/+
+/+
+/+
+/+
…. we still don’t
know in the F2!
+/+
+/+
+/+
Meanwhile
+/ a
+/+
+/ a
+/+
The problem with diploids in hunting for new recessive mutations:
-- if we can’t self.
mutagenize
Female X Male
+/+
+/+
+/+
+/b
X+/+
+/+
(must keep
populations
separate!)
+/+
+/+
+/+
+/+
+/+
Mate
inter se
at best
+/+
+/ a
…. we still don’t
know in the F2!
F1
X+/+
F2
..but at least now we have
potential mates with a-
+/+
+/ a
+/+
+/ a
male?
+/+
female?
at least some chance:
Mate
inter se
a /a
The problem with diploids in hunting for new recessive mutations:
-- if we can’t self.
mutagenize
Female X Male
+/+
+/+
+/+
+/b
+/+
F1
+/ a
X +/+
+/ a
+/+
+/+
+/+
+/+
+/+
Mate
inter se
only +/+
F2
+/+
+/ a
+/+
+/+
if we cross them,
a-/a- will come:
can we
do better
than
mating
inter se?
a /a
It would help if we could keep track of chromosomes:
mutagenize
a- = mutagenized chromosome with new mut.
+ = mutagenized but not desired mutant
+ = non-mutagenized from original Mom
+ = non-mutagenized from F1 mate
Female X Male
+/+
+/+
+/+
+/b
+/+
F1
+/ a
X +/+
+/ a
Even nicer
if we could
eliminate
extraneous animals
F2
+/+
+/ a
+/+
+/+
if we cross them,
a-/a- will come:
we can
do better
than
mating
inter se
a /a
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