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Fly Genetics (fall 2012)
Pat O’Farrell [email protected] - 6-4707
Lecture 4 Pseudoalles – Noncoding “genes” – homeotic transformation and
regulation – genetic memory and the polycomb system.
Sensitized genetics – a powerful screen for modifiers
Psuedoalleles
Homeotics and the special heritage we have in fly genetics
Polycomb and trithorax class mutations, the genetics of chromatin states
Pairing and transvection
Biological/Developmental/Genomic issue
Greater than 90% of our DNA does not code for gene products and appears to have regulatory
roles. We do not know how it works. We do not know the logic of its evolutionary conservation.
We suspect that differences in these sequences distinguish species. Can genetics lead us to
answers?
General reading:
New: Two nice papers from Ian Duncan summarize the seminal contributions of Ed Lewis to
genetics and biology. They can found at
http://www.genetics.org/cgi/content/full/160/4/1265
http://www.genetics.org/cgi/content/full/161/1/1
I’ve referenced it before, but in case you didn't get what I said or want a repeat with a different
perspective, try this. It is a review that beautifully represents many of the things that I have tried to
teach. St Johnsoton, D. (2002) The art and design of genetic screens: Drosophila melanogaster.
Nature Rev. Genet. 3, 176 -188
Sensitized Genetics
A new a powerful way for screening for genes that contribute to particular biological function. This
approach was first developed and used by Mike Simon, an ex-student of our department, when a
post-doc in Gerry Rubin's lab.
Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM (1991) Ras1 and a putative guanine
nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine
kinase. Cell 67, 701-16
Idea based on several facts.
1. One good copy of almost any region in the genome is sufficient for wt function (demonstrated
by small deletions genome wide Lindsley et al., 1972) (haploinsufficiency is rare)
2. Removing one copy of a gene reduces its function in half (Muller et al., 1931) (Surprising fact
that has verified repeatedly and is true for the vast majority of genes).
3. When an essential pathway has been compromised by a hypomorphic mutation so that a weak
phenotype is seen – the severity of this phenotype is very sensitive to conditions.
It was argued that a hypomorphic mutant would reduce the activity of the entire pathway to which it
contributed and consequently reducing the dose of another gene in the pathway might further
reduce the activity of the pathway and exacerbate the phenotype, while the same heterozygous
defect would have no effect in the absence of the sensitizing mutation. The authors studied
receptor tyrosine kinases that are required for normal eye development and discovered Ras and
Ras regulatory proteins as enhancers of defects in RTK function and suppressors of mild gain-offunction RTK mutant. Their work ended up organizing the previously chaotic field of Ras signaling,
and revealed powerful and general way to screen for genes in particular functional pathway.
WT eye
Ellipse (gain of function RTK)
Ellipse, modifier mutant/+
Almost any process in molecular biology, cell biology, or development can be dissected by modifier screens
in the eye (note the eye is best but similar things can be done in other tissues). To do this one might express
an RNAi in the eye that compromised a particular process, and then cross to introduce mutagenized
chromosomes and identify modifiers of the phenotype. Note that these show up as dominants even if they
are effectively recessive genes. As a dominant screen, there is no requirement to homozygous the mutation.
Individual F1 progeny are screened and the mutants subsequently recovered. Many of the mutants are
recessive lethals – explain this to yourself.
A special heritage
http://www.genetics.org/cgi/content/full/160/4/1265
http://www.genetics.org/cgi/content/full/161/1/1
The work of EB Lewis beginning in the 1940’s gave us an extraordinary view of genetic and of
development. The scientific community is still trying to catch up with the implications of some of
the things he found. I will mention two genetic phenomena that he described because they are
especially meaningful to the genetics of regulation.
Homeotic mutations: Cause the development of normal structure in the wrong place!
(The normal function of a homeotic gene is to direct the expression of the normal structure in its normal place. Homeosis
(the transformation of one structure into another) occurs when a homeotic gene is ectopically expressed in a position
where it should not be, as in the neomorphic Antp mutant, or indirectly because of the absence of expression of a
homeotic gene in its normal domain. Regulatory interactions (repression hierarchy) among the homeotic genes
contributes to their normal patterns of expression, and loss of expression of one gene can result in another gene being
derepressed in the formally occupied domain.)
Lewis studied a cluster of “genes” specifying the differences between segments, the
Bithorax Complex.
Fig. Phenotypes of
bithorax complex
mutations. (A) Dorsal
view of a wild-type male.
The T2 segment
produces the single pair
of wings as well as
almost all of the dorsal
thorax. Dorsally, T3
produces only the
halteres, small clubshaped organs located
posterior to the wings.
(B) The famous fourwinged fly, in which T3
is transformed to T2.
This male is hemizygous
for the triple-mutant
combination abx bx3 pbx. The abx pseudoallele has effects similar to bx mutations, but causes a stronger transformation
of the very anterior portion of T3. (C) A Cbx male showing transformation of T2 toward T3. Generally, Cbx transforms
only the posterior portion of T2, as seen on the right side of this fly. Occasionally, all of T2 is affected, as occurs on the
left side. (D) Ventral view of a wild-type female. Each of the thoracic segments produces a pair of legs. (E) Ventral view
of a bxd hemizygous female. An extra pair of legs is present as a result of the transformation of A1 toward T3. (F) Ventral
view of an Hab female. The third pair of legs is lost because T3 is transformed toward A2. (B–F courtesy of E. B. Lewis.)
Pseudoallelism:
Ubx/bx – transformed the anterior half of the haltere to wing
Ubx/pbx – Transfromed the posterior part of haltere to wing
bx/pbx – wild type
Logic of complementation places bx and pbx in the Ubx complementation group yet they
complement each other. What is the explanation?
Transvection:
Ubx/bx – mild phenotype
Ubx + R/bx – strong phenotype
Ubx + R/bx + R – Mild phenotype
R is rearrangement (and inversion) were the break points are far away from the Bithorax Complex.
The rearrangement interfered with alignment/pairing of the homologs when it was heterozygous,
and this increased the severity of the phenotype. Why?
The Bithorax Complex (BxC)
The Complex is huge, and very little of sequence is occupied by coding sequence. There are three
homeobox transcription factors, and about 100kb of regulatory sequence. Molecular dissection of
this and other loci have taught us that there are blocks of regulatory sequence that are about gene
size, that guide spatial and temporal aspects of the program of gene expression. Mutations such
as bx and pbx appear to be mutations in such regulatory sequences. Now can you explain
pseudoallelism?
For the future. Can genetics dissect the logic of regulation, the task that the bulk of our
sequences are devoted to?