Tools P element – a versatile transposon.

P-element structure Figure 14-16

Types of vectors 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: 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 Position independent expression Cis acting sequences GFP reporter + + + +  -

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.

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