Transcript lecture 2

Tools
• P element – a versatile transposon.
• P-element structure
Figure 14-16
Types of vectors
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General transformation.
Reporter vectors.
Regulated expression vectors.
Gateway cloning system vectors.
FRT containing vectors.
φC31 site specific recombination vectors.
Fold back RNA vectors.
Transposon mutagenesis
• Many variants:
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Simple
Enhancer traps
Fusion protein
Over/ectopic expression
A screen using the P element as a
mutagen.
w; P[w+lacW] X w;TMS, Δ23 Sb/Dr
w; P[w+lacW]/TMS, Δ23 Sb X fz th st in
Look for flies that are phenotypically fz or in.
These are likely to be due to a P insertion into fz
or in.
P as a mutagen
• How to test if a mutation is due to a P
insertion.
• Can Δ23 induce reversion? Frequency
usually 30-.1%.
• Reversion is often imprecise – can lead to
deletion.
Reversion of a P insertion (in this case P carries
w+):
w; fryP/TM6 X TMS, 23, Sb/Dr
w; fryP/TMS, 23, Sb X w; TM6/DcxF
Screen for white eyed flies with normal bristles.
These will be w; fryRV/TM6 (or w; fryRV/DcxF) .
Establish a stock that is w; fryRV/TM6 and test
for fry function.
Screen fry- chromosomes molecularly to identify
cases where imprecise excision lead to a deletion
of part or all of fry.
Assay of cis acting regulatory sequences
Cis acting sequences
GFP reporter
Position
independent
expression
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figure 5.16
Tool
• Collection of transposon insertions.
• Goal: an insertion in every gene.
Gal4
Galactose
Gal80
UAS
No galactose – Gal4 does
not activate transcription
In presence of Galactose,
Gal80 does not bind Gal4,
GAL4 activates transcription
Gal4 enhancer trap insertions provide a wide range of cell
type/tissue pattern/developmental stage specific gene
expression drivers.
figure 5.17
A temperature sensitive GAL80 transgene is available
that can be used to temporally control GAL4.
Genetic Mosaics
• Provides a cell marker that cannot be
diluted out. Very valuable for tracing cell
lineage.
• Can use to study gene function.
– Gets around some aspects of pleiotropy.
– Allows additional functional tests of genes and
pathways.
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• Autonomous vs non-autonomous
Lineage Restrictions
Log
of
clone
size
Log of
clone
frequency
Developmental Time
Lineage Restrictions
• Early attempts to identify lineage
restrictions were hampered by the difficulty
in obtaining large clones.
• Data did show that clone shape was not
random. For example, clones on the leg are
long and thin (extended along the proximal
distal axis.
Lineage restrictions
• How to get around the clone size vs clone
frequency problem.
• Minute technique.
Lineage Restrictions
• Anterior - Posterior Compartment boundary.
• Also a compartment for the expression of
regulatory genes.
• E.g. hedgehog and DPP are expressed along
AP boundary.
Ways to generate clones that
express a gene that neighboring
cells do not.
• Flip out
FRT
FRT
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P
act 5C
promoter
Poly A
Poly A
Gene of interest
Flip Out
P