Fly Genetics (fall 2011)
Pat O’Farrell ofarrell@cgl.ucsf.edu - 6-4707
Lecture 3 How genes function in space and time & Intro to the “Genetic tool box”
Genetics concepts new to this section:
P-element tranformation – the fly version of transformation.
The GAL4/UAS two component expression system.
Using FLP mediated site specific transformation at FRTs – clonal lose and gain of function.
Positive marking of clones.
RNAi – the fly version
Developmental
Principle: Cells whose fate is determined produce clones that are limited to specific tissues/areas.
Reciprocally, the distribution of daughter cells reveals the developmental potential of an earlier cell.
General reading:
This is very lucid description of key developmental concepts highlighted by clonal analysis in
Drosophila. The pdf is on line and it is recommended reading.
Crick FH, Lawrence PA. (1975)
Compartments and polyclones in insect development. Science 189, 340-7
Development of P element mediated transformation: Rubin GM and Spradling AC (1982) Genetic
Transformation of Drosophila with Transposable Element Vectors. Science 218, 348-353
The single hop insertational mutagenesis strategy: Cooley, L., Kelley, R., and Spradling A. (1988)
Insertional mutatgenesis of the Drosophila genome with single P elements. Science 239, 1121- 8
This now dated review still provides a background on many of the diverse tools that have been
developed on the basis of regulated expression of transgenes by the GAL4 transcription factor.
Duffy, JB (2002) GAL4 System in Drosophila: A Fly Geneticist;s Swiss Army Knife. Genesis 34, 115
A technical paper that gives an excellent description of the process of producing positively marked
clones, which can also be used for ectopic expression of genes in specific cells and for RNAi
knockdown in specific cells.
Wu, JS and Luo L (2006) A protocol for mosaic analysis with
a repressible cell marker (MARCM) in Drosophila. Nature Protocols 1, 2583
Hybrid dysgenesis
!
Fly from the
wild
!
X
Few progeny
The progeny have shriveled gonads and are almost sterile
The few F2 progeny sport frequent mutations
Dysgenesis is due to Infection by Transposition Element
Transposition elements: There are three modes of transposition,
conservative (or cut and paste), replicative and retrotransposition. The P
element uses cut-and-paste, catalyzed by transposase.
Regulation –
Repressor of transposase in the eggs of wild flies
Repressor – small RNA based but not the type you are used to.
Three types:
RNAi
Micro RNA
PIWI RNAs
Fly from the
lab
P element as a
Transgenesis Tool
Transposase source:
Delta 2-3 on III markedwith Kinked
(Ki).
P element vectors: Cis sites for
transposition, marked with a w+ gene,
refined cloning expression tools.
Insertion of DNA: Transposon is
introduced by injection into
transposase expressing egg and
DNA hops from plasmid into
genome. Events are detected by
the introduction of the w+.
Commercial generation of
transgeneics for ~ $200.
Mobilizing a P – to mutagenize/trap etc.
Cross flies carrying a w+ P element to flies expressing transposase
Male progeny with both element and enzyme --- get transposition
Cross and recover progeny without the transposase, and with w+ on different chromosome than it
started on.
P element mutagenesis – a gene knockout collection:.
Genes and insertions in 100 kb of genome (see FlyBase)
.
Enhancer trapping:
An enhancer trap marks dentridritic
projections of specific sensory cells in the epidermis of
the larvae (right).
GAL4 System/2 component systems
GAL4 Drivers:
Enhancer traps that drive GAL4 expression.
UAS targets:
Genes of interest under the control of a UASGAL4.
Added control:
GAL80 inhibits GAL4. Removing GAL80 (e.g. by mitotic recombination) allows GAL4 to function
only in the cells that lack GAL80 – e.g. in the clone. Using temperature sensitive GAL80, a GAL4
system can be held repressed at low temperature until a desired time when a temp upshift
inactivates the inhibitor and turns on transcription.
FLP/FRT System
The yeast FLP recombinase acts at FRT sites to induce recombination. This activity has been put
to great use in flies.
FLP based mitotic recombination to produce clones:
FLP can be used in various ways:
Flip out cassette – Clonal expression
FLP is also useful in different strategy in which one induces clonal expression and/or loss of
expression of transgene.
A Little about the fly eye
.
Making the eye homozygous for one chromosome arm
http://www.BruinFly.ucla.edu/methods.php
Also see - Stowers RS, et al. (2002) Axonal transport of mitochondria to synapses depends on
milton, a novel Drosophila protein. Neuron. 36(6):1063-7
Positively Marked Clones (MARCM)
Recessive markers only mark homozygous cells produced by recombination, but GFP marks both
UAS-GFP heterozygous and homozygous cells. Consequently, to mark clones with GFP,
investigators used to follow loss of GFP in the homozygous loss clone. It was difficult to follow
small clones in a sea of positive signal. GAL80 allows positive emarking.
Second two component system
(from Quinic acid regulon of Neurospora crasa):
Q system (C.J. Potter and L. Luo)
Independent TF,
repressor and sites
Combine with GAL4 to make twin spot MARCM
RNAi in flies
Long dsRNA
Flies
works
Worms
works
Cell autonomy
autonomous
Transitive/*
intransitive
Amplification
Intransitive *
Spreads throughout
(except nervous system)
Transitive
Apparently not
Amplifies signal



Mammals
Doesn’t work: induces interferon
response that nonspecifically
shuts down translation (viral
defense strategy)
?
?
?
Transitive:
Intransitive:
Importance: Control.
RNAi in cell culture
Cell culture RNAi screening in Drosophila cell culture is uniquely simple.
Advantages of Drosophila S2 cells:
1. Take up long dsRNA directly from media
2. Robotic PCR from genomic DNA, & symmetric transcription -> dsRNAs to all of the genes.
3. The whole genome can be screened days.
4. Experimental dissection. One, two, three or more dsRNAs can be used to knockout
combinations of genes, and film (e.g. cytokinesis defect).
Echard, A., Hickson, GRX, Foley, E., and O’Farrell, PH. (2004) Terminal Cytokinesis Events Uncovered after an RNAi
Screen. Curr. Biol. 14(18):1685-93
Foldback transgenes
http://www.vdrc.at/rnai-library/
Two libraries –1. The GD Library:
2. The second library has inserts (10,714) in a common site. Similar except a new transgenic
technology that uses a phage site specific recombinase (phiC31) to insert RNAi constructs at
specific docking sites. The benefit is that the context effect on gene expression is removed.
Disadvantage – can’t be combined in large numbers because they are allelic.
2 other large libraries of RNAi constructs – one in Boston and one in Kyoto. Boston also
provides reagents and technical help for cell based RNAi screens
RNAi knockdowns – in space and time
Since RNAi transgenes are under the control of UAS, they can be expressed ubiquitously or in any
tissue with the appropriate driver and they can be regulated by GAL80ts for temporal control, or by
GAL80 in the MARCM system to specifically inactivate the gene in individual marked cells.
Using gene dose, and temperature the severity of knockdown can be adjusted.
RNAi by injection
dsRNA can be injected into the early embryo and maternal RNAs can be eliminated or
accumulation of zygotic product blocked – maternally provided proteins are of course immune to
this.
Triple RNAi of the three mitotic cyclins of Drosophila by dsRNA injection into the anterior (left) pole
blocks subsequent mitoses and the domain of action expands with time to halt successive waves
of mitosis.
McCleland ML and O’Farrell, PH (2008) RNAi of Mitotic Cyclins in Drosophila Uncouples the Nuclear and Centrosome
Cycle. Curr Biol. 18(4):245-54
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?