Download bYTEBoss 140-S08

Important piece of information for Monday:
Testing site:
Last names beginning A-L: 101 Barker
Last names beginning M-Z: 240 Mulford
(1) Deletion mapping of genes (Benzer)
using the segregational test for allelism:
Can you recover a wildtype allele
from the mutant hybrid?
(a recombination test)
If you can, the point mutant is outside the region deleted.
(2) Deletion mapping of genes (Bridges)
using the functional test for allelism:
Is the mutant hybrid phenotype wildtype?
(a complementation test)
If it is, the point mutant is outside the region deleted.
Deletion mapping based on a complementation test
w- rst- fa- = w- (mutant) phenotype (failure to complement)
Df(1)256-45) hence white is within region deleted.
phenotype
Bulge in the synapsed
polytene chromosomes
shows what is deleted.
Fig. 14.8
Inversions also have helped locate genes on the Drosophila
polytene chromosome map via complementation tests
may knock out bw+
…cause loss of ability to complement bw-
Inversion breakpoints
bw+
In(3R)bw-
In(3R)bw-/bw- = brown eye phenotype
The complementation test
as an operational definition of the gene
is not quite as straightforward
as it may sound.
genes can have very
complex complementation patterns
because of all the various kinds of information they contain
that work only in cis
(and all the various ways in which that information
can be changed by mutation)
From your text:
p227 Heading: “rII regon has two genes”
Is this statement compatible with the statement that
complementation groups are what we want to call genes?
(starting bottom of p291):
A gene is not simply the DNA that is transcribed into the mRNA codons
specifying the amino acids of a particular polypeptide. Rather, a gene is
all the DNA sequences needed (IN CIS) for expression of the gene into
a polypeptide product. A gene therefore includes the promoter
sequences that govern where transcription begins and, at the opposite
end, signals for the termination of transcription. A gene also includes
sequences dictating where translation starts and stops. In addition to all
these features, eukaryotic genes contain introns that are spliced out of
the primary transcript to make the mature mRNA. Because of introns,
most eukaryotic genes are much larger than prokaryotic genes.
So promoters are part of genes.
Are promoter mutations part of complementation groups?
How about intron mutations?
A bacterial promoter
(cis-acting information for
transcription start)
51 bp
Fig. 8.12
transcript--->
the fine-structure map of rIIA & rIIB generated by
recombination between
mutants in the same genes (as complementation groups)?
1,612 INDEPENDENT mutants mapped for Fig. 7.21 (and ultimately >3000)
B -- A
There are 12 bp between
rIIA & rIIB “genes”
(T4 has 168,903 total bp)
…hence no room for a standard promoter
rII makes a polycistronic mRNA
P
rII-A
rII-B
Figure 17.5: The Lactose Operon in E. coli
classic example of
polycistronic mRNA
rII “complementation map” of point mutants
(a very different kind of “map”)
7
23
rIIA&B mutants in the promotor for a polycistronic mRNA
rII-A
3
5
66
71
rII-B
2
65
87
99
three complementation groups?
No, one complex complementation group
(one “gene”) provided 7 and 23 don't meiotically map as large deletions
Eukaryotic genes
are even messier:
Alternative pre-mRNA splicing
is what allows Drosophila
DSCAM gene to make
30,000 different proteins
6 vs. 7-8 alternative
exons
introns
…and the
regulatory regions
(non-protein-coding)
can extend enormous
distances on both sides
exons
Fig. 8.18
(NATURE 184:1927-29, 1959)
largest class
(r+ encodes a single polypeptide with three sequential enzymatic activities
required for purine biosynthesis)
These are
the mutants
that argue for
one gene with
a complex
complementation pattern
(14/31)
This “map” of rudamentary alleles
does not imply anything about where
the various mutations might lie on a meiotic map.
It is simply a schematic representation of a collection of data from
a series of complementation tests
designed to determine functional allelism
Let’s consider three r mutant alleles:
did they complement (Y or N)?
a -/b - : Yes
they don’t overlap
on the map
…and the hybrid fly looked: wildtype
did they complement (Y or N)?
a -/h - : No
they do overlap
on the map
Consider a new r mutant allele, rz3
z3 / a
z3 / h
z3 / i
Which heteroallelic combination (hybrid) is
most likely to have a mutant phenotype?
z3 / i
Which heteroallelic combination is most likely to
generate a wildtype allele during meiosis?
? no basis for
a determination
This “map” tells us next to nothing about the
possible molecular basis
for the complex complementation pattern
…but so long as we have reason to believe
that group i contains at least some point mutants,
all the mutants on this “map” are likely to be in the
same (thing that we want to call a) gene.
…and i is the most
frequent class
Mutations (changes in DNA):
the lifeblood of genetic analysis
(1) What kinds can we make? (categories)
(2) How do we make them? (mutagenesis)
(3) How do we find them? (mutant screens & selections)
(4) Why bother?
Central Dogma:
information flow
N.A. Protein
?
the return
of the fly
The young S. Benzer: (1950s)
phage T4
as his genetic workhorse
(while fly work was in its
"eclipse period")
Benzer's question for the fly:
how do genes encode
complex nervous systems?
An older Benzer: (1967-2007)
fruit fly (D.melanogaster)
as his genetic workhorse
•founded many of the most
interesting areas of modern
behavioral genetics
(clocks & learning, etc.)
•set up the experimental system most
effective for studying fly development:
the compound eye
Can mutations affecting the fly eye tell us anything about our eyes?
Fig. 20.10
hypomorphic or null mutations hypomorphic or null mutations
of the eyeless gene in an adult
of the Pax-6 gen
fruit fly
In a fetal mouse
(homozygote would have
(human aniridia
died as an embryo) Fig. 20.4
dominant “genetic disease”)
Induced “ectopic” expression
of mouse Pax-6 gene product
Fig. 20.10
Fig. 8.31, panel d:
A neomorphic dominant mutation in the fly Antennapedia gene
causes ectopic expression of a leg-determining gene
in structures than normally produce antennae
(actually its wildtype function is to force cells
that would otherwise make an antenna to make a leg instead)
and abx
Fig. 20.23
…but the most informative mutations may not
be compatable with survival to the adult stage
(a somewhat revolutionary idea, remarkably enough)
Fig. 20.2
a fruit fly 9h after fertilization
head
tail
wildtype (ftz+)
ftz amorph (null mutant) homozygote
(it’s recessive lethal)
something Thomas Hunt Morgan never guessed (among other things):
you can get a huge amount of phenotypic information out of
the skin of a dead maggot
by 16h after fertilization, even if doomed
denticle belt pattern on the larval cuticle
(cuticle: “skin,” tho. actually “skeleton”)
(denticles: maggot tire treads)
reveals where fly cells think they are in space
Wieschaus and Nüsslein-Volhard, Nobel Prize 1995
Fig. 20.19
maximally informative mutant phenotypes
for understanding metazoan pattern formation
Krüppel
wildtype
hunchback
knirps
(ftz is in the “pair rule” family, not the “gap” family of mutant phenotypes)