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1

DNAse hypersensitivity assay (finding protein-binding control elements) / Molecular Probes

- Ethidium bromide (EtBr) allows detection of DNA or RNA fragments in gels, but is not specific to any particular nucleotide sequence.
- All DNA bands will stain with EtBr.
- In contrast: "labeled" DNA fragments can be used to identify specific sequences within whole genomes.

2

DNAsel hypersensitivity

-You want to detect this one particular region in the genome:
- Chromatin from nuclei (whole genome)
- Treat with increasing concentrations of DNAsel.
- Remove proteins; isolate DAN ( whole genome)
- Digest with EcoRI
- Hundreds of thousands or EcoRI fragments per whole genome per single cell.

3

Hybridization

- Single stranded nucleic acid probes (usually double-stranded but strands are separated by heat and then diluted to avoid re-annealing) are used to detect RNA or DNA molecules in Northern or Southern blots, respectively.
- The pairing of complementary sequences between the probe and target nucleic acid is called hybridization.

4

Use Gal4/Gal80/UASg system to manipulate gene expression in flies

- Twelve cells that differentiate into neurons or glia later in development. Want to label subsets with GFP and follow their path during development.
- 4 genes expressed in subsets of these cells. Find regulatory sequences driving 4 genes' expression.

5

Experimental Approach

- Make 4 constructs with 4 different regulatory sequences driving Gal4 expression (flies don't express either Gal 4 or Gal80).
- Introduce each construct into fly embryo along with GFP gene under the control of UASg.
- If Reg2 binds positive TFs in a given cell, GAL4 gene will become active and produce GAL4.

6

To achieve this goal, make 4 more gene constructs:

- Make 4 constructs with 4 different regulatory sequences driving Gal80 expression (flies don't express either Gal4 and Gal80)
-If positive TF's bind Reg2 in cell, Gal4 will be produced BUT, if positive TF's ALSO bind Reg1 in cell, GAL8 will be produced.
- GAL4 cannot bind UAS -> no GFP produced.