Transcript Document

The date for the review for the midterm has been changed to
Thursday eve. (7-9p) April 10
since there is a conflict with a night exam on the 9th.
I’ll announce where the session
will be held as soon as I know myself.
Reading on sex:
pp85-88 (In many species…)
pp109-110 review (Analysis of rare mistakes…)
pp516-518 (Changes in chrom. # --to--Some euploid…)
Table 14.2 (p526)
pp664-665 (RNA splicing helps regulate gene expression)
pp669-676 except 670-71 (Sex determination in Drosophila…)
On Monday:
(1) genetically sensitize the system:
“turn” lof recessives into dominants (but only with respect to
one non-essential aspect of their function)
Poising the activity level of “your favorite gene”
on a phenotypic threshold to make other genes
that work with it in a regulatory pathway
haploinsufficient
…but only with respect to the functioning of “your favorite gene.”
So that:
(1) we can identify new mutations of interest in the F1 generation
(first generation after mutagenizing the parents)
AND
(2) can overcome some complications of pleiotropy
…so that we can more easily study the non-vital aspects of the
functioning of genes that ALSO have vital functions
(1) genetically sensitize the system:
“turn” lof recessives into dominants (but only with respect to
one non-essential aspect of their function)
Based on the rationale that:
Wildtype organism must normally have an excess of most genes’ activity
as insurance against fluctuations in
the levels of activity of various genes in a pathway during development
…if take away that cushion for any one gene in a pathway,
now make the normal operation of the pathway
…with respect to that one gene…
more vulnerable
to reductions in the levels of other gene products
with which it works
Another way around the limitations of pleiotropy in genetic screens:
(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
without making them dominant !!!
In your text's glossary:
genetic mosaics
see: mosaics
“an organism containing tissues of different genotypes”
cells
…all derived from a single initial cell (zygote)
genetic mosaic vs:
organism
(genetic) chimera: an embryo or animal composed of cells
from two or more different organisms
individuals
genetic mosaics are extremely useful for determining
where a particular gene’s function is needed:
Your text short-changes the key concept
of cell autonomy in gene function
Fig 20.14:
+
+
-
+
+
genetic mosaics are extremely useful for determining
where a particular gene’s function is needed:
nonmosaic
wildtype
L2
L1
L2
L1
phenotypic
effect of
ag- vs. ag+
differentiation
non-mosaic
ag- mutant
L2
L1
L2
L1
In what cell type (L2 vs. L1) is ag+ function needed
to elicit normal differentiation (
) of L1?
Where is
ag+ needed?
mosaic
white
marks ag-
nonmosaic
wildtype
ag-
ag+
mosaic
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
ag+
non-mosaic
ag- mutant
L2
L1
ag-
L1 differentiates
abnormally despite
being ag+
L1 differentiates
normally despite
being ag-
ag seems to work in L2 to signal L1 (like boss)
For ag, the cell whose phenotype is mutant is not the cell whose genotype
is mutant. Hence ag is not cell autonomous with respect to the L1 phenotype
The “genetic marker” is cell autonomous (that’s why it was chosen),since the
cell whose marker phenotype is mutant is the cell whose marker genotype is mutant.
…if a cell’s genotype in the mosaic dictates that cell’s phenotype
nonmosaic
wildtype
ag-
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
L2
L1
ag+
non-mosaic
ag- mutant
ag is
nonautonomous
ag+
ag+
ag-
ag-
ag seems to work in L2 to signal L1
When discussing autonomy/nonautonomy,
need to specify the phenotype in question:
…ag is non-autonomous with respect to the L1 differentiation phenotype
…perhaps ag is autonomous with respect to generating the signal in L2.
(if we had an assay for the signal, we might see that phenotype directly)
T.H.Morgan published on genetic mosaics in 1914:
do males normally
have white eyes?
X
X
X
X
first nuclear
division of an
XX zygote
Hence,
~50%
w+ vs. w…mosaic patches
too large
for
our purposes
X
X
w-/w+
w-/--
XX
XO
XO XX
otherwise
diploid
(AA)
stable thereafter
Fig. 14.33
Another way around the limitations of pleiotropy in genetic screens:
(2) use genetic mosaics to screen for recessives in the F1
…look for homozygous mutant clones
in otherwise heterozygous animals
…identify (and recover) new recessives in the F1
even works for new mutants that are recessive lethal or sterile
provided
-- one generates clones in only a small fraction of all cells
or
-- one generates clones only in non-essential tissues
Another way around the limitations of pleiotropy in genetic screens:
(2) use genetic mosaics to screen for recessives in the F1
…look for homozygous mutant clones
in otherwise heterozygous animals
Based on a phenominon discovered (‘30s) by former chair
of U.C. Zoology Dept: mitotic recombination
but improved upon enormously in modern times
…only possible because of a very strange aspect of
fly chromosome behavior:
homologous chromosomes pair during mitotic interphase
Stern’s observation:
…for fly heterozygous for recessive
cell-autonomous l.o.f. alleles
of two genes:
yellow(body)- singed(bristles)+
yellow(body)+ singed(bristles)-
If irradiated (ionizing) during development
occasionally saw
odd ADULT fly:
deduced:
mistake in mitosis,
not new mutant alleles
most flies
wildtype
patch of
y/y ?
patch of
sn/sn ?
“twin spot”
Fig. 5.23 p152
Consider what it is about mitosis that insures daughter
cells will have the same genotype as their mother cell:
XmXp
y- sn+
y+ sn-
y-
sn+
m
y-
p
y+ sny+ sn-
then:
if instead:
y- sn+
y+ sn-
y-
sn+
y+ sn-
sn+
m
m
y- sn+
y- sn+
p
p
y+ sny+ sn-
m
p
m
p
=
m
m
p
p
y- sn+
y+ sn-
m
y- sn+
y+ sn-
m
p
p
What if DNA breaks and improper repair after S phase
change relationship between genes and centromeres:
y- sn+
y+ sn-
y-
sn+
m
y-
p
y+ sny+ sn-
sn+
m
m
mistake
p
p
NO CHANGE if instead:
yellow
y- sn+
y+ sny- sn+
y+ sn-
m
m
m
m
p
p
y- sn+
y- sn+
m
y+ sny+ sn-
m
p
twin-spot
singed
p
DNA breaks and improper repair after S phase
generate the “twin-spot” of cells homozygous for y and sn
y- sn+
m
y+ sn-
p
y- sn+
y- sn+
y+ sny+ sn-
twin-spot: because progeny
of the y/y & sn/sn original
cells tend to stay together
ADULT
EXTERIOR
size of patches depends on when
abberrant mitosis occurs
m
m
mistake
p
p
y- sn+
y+ sn-
y- sn+
y+ snGrowth
m
m
m
m
p
p
m
y/y (yellow)
y- sn+
y- sn+
patch (clone) of
sn/sn (singed)
y+ sny+ sn-
m
patch (clone) of
p
p
Fig. 5.24 (p152)
What if we had induced a NEW recessive MUTATION on a mutagenized
y- sn+ chromosome (in the father’s sperm) that affected cell growth parameters?
y- m- sn+
y+ m+sn-
y-
m-
sn+
p
y- m- sn+
m
y+ m+ sny+ m+sn-
p
p
mistake
m
m
y- m- sn+
p
m
+
y m+ sn- m
p
y- m- sn+
m
y+ m+ sn- pp
m
zygote
m-/mm-/m+
odd patch of
y/y yellow
patch of
sn/sn
singed
y- m- sn+
y- m- sn+
p
y+ m+sny+ m+ sn-
p
m
m
..appearance of an ABNORMAL homozygous yellow patch next to homozygous
singed patch would SIGNAL that the female carried an interesting
new mutant allele
y- m- sn+
y+ m+sn-
y-
m-
sn+
p
y- m- sn+
m
y+ m+ sny+ m+sn-
p
p
mistake
m
m
y- m- sn+
p
m
+
y m+ sn- m
p
y- m- sn+
m
y+ m+ sn- pp
m
zygote
m-/mm-/m+
odd patch of
y/y yellow
patch of
sn/sn
singed
y- m- sn+
y- m- sn+
p
y+ m+sny+ m+ sn-
p
m
m
..and such a female would be fully viable
even if
the new recessive mutant allele would have been lethal
(perhaps even embryonic lethal -- killing long before adult stage)
if a significant fraction of her cells had been homozygous for it.
odd patch of
y/y yellow
m-/mm-/m+
patch of
sn/sn singed
ADULT
EXTERIOR
hence
avoids complications
of pleiotropy
…lethal
either because of a defect
in the same function
affecting adult cuticle, or because
of a defect in some other process
Problems that had limited the use of
radiation-induced mitotic recombination
for genetic screens:
-- mitotic recombination infrequent
-- position of exchange not controlled
-- radiation used to induce is damaging
-- tissues in which occurs are not controlled
Solution:
induce using site-specific yeast recombination system
FLP: recombinase (protein that catalyzes recombination at FRTs)
FRT: DNA target (34 bp) site for recombinase
FRT site
y- sn+
y+ sn-
y-
sn+
m
y-
p
y+ sny+ sn-
source
of FLPase
…but only induced
when & where
FLPase induced
sn+
m
m
p
p
targeted “mistake”
yellow
y- sn+
y+ sny- sn+
y+ sn-
m
m
m
m
p
p
y- sn+
y- sn+
m
y+ sny+ sn-
m
p
twin-spot
e.g. eye precursor cells
singed
p
lats/lats clone in eye l(3)93B/l(3)93B clone in eye
of lats/+ adult
of l(3)93B/+ adult
(“large tumor suppressor”)
3rd instar larvae
left: wildtype
right: lats/lats
(doomed)
(Xu et al, Develop.121:1053 1995)
homozygous lats clone on thorax of lats/+ adult fly
(Xu et al, Develop.121:1053 1995)
Genetic mosaics have zillions of uses
besides just facilitating mutant isolation
…and geneticists have ways of controlling
exactly when and where FLPase is generated
…and hence
exactly when and where mitotic recombination is induced